Therapeutics using adipose cells and cell secretions

ABSTRACT

The invention relates to compositions comprising (i) adipose tissue-derived cell secretions or (ii) an adipose tissue-derived cell suspension, optionally comprising adipocytes, or (iii) a combination of adipose tissue-derived cell secretions and an adipose tissue-derived cell suspension, optionally comprising adipocytes, and to their use in pharmaceutical compositions and methods for treatment of various conditions. The invention also relates to improved methods, agents and compositions for cryopreservation of cells.

RELATED APPLICATIONS

The present application is a US national phase of InternationalApplication No. PCT/AU2012/001140 filed on Sep. 21, 2012, which claimsbenefit from Australian provisional patent application No. 2011903938entitled “Therapeutic methods and compositions” filed on 23 Sep. 2011and from Australian provisional patent application No. 2012901350entitled “Therapeutic methods and compositions” filed on 4 Apr. 2012 andfrom Australian provisional patent application No. 2012903646 entitled“Therapeutic methods and compositions” filed on 23 Aug. 2012, the entirecontents of each of which is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to compositions comprising (i) adiposetissue-derived cell secretions or (ii) an adipose tissue-derived cellsuspension, optionally comprising adipocytes or (iii) a combination ofadipose tissue-derived cell secretions and an adipose tissue-derivedcell suspension, optionally comprising adipocytes, and to their use inpharmaceutical compositions and methods for treatment of variousconditions such as inflammatory disorders, ligament injuries, tendoninjuries, or for alleviating pain associated with such conditions, aninflammatory disorder, a ligament injury or a tendon injury, in amammalian subject. In the methods of the invention the compositions areadministered to a site on a patient remote to the site affected by theinflammatory disorder or condition. The invention also relates to theuse of such compositions in pharmaceutical compositions and methods forthe treatment or prevention of disease in an intensively farmed animal,wherein said administration is by subcutaneous or intramuscularinjection. The invention also relates to the treatment of pain in asubject, the treatment being by subcutaneous injection or intramuscularinjection. The invention also relates to treatment of neuropathic painin a subject. The invention also relates to improved methods, agents andcompositions for cryopreservation of cells.

BACKGROUND OF THE INVENTION

Adipose tissue contains a cell population of large lipid filledadipocytes, and a non-adipocyte cell population, which comprises cellsassociated with various connective fibres and cells associated withcapillaries and larger blood vessels. The non-adipocyte cell populationis also thought to comprise a population of adipose-derived adult stemcells and consequently there has been interest in using adipose tissueas a source of isolated stem cells for various therapeutic applications.

In general, methods for obtaining adipose tissue derived presumptivestem cells involves depleting adipocytes from adipose-derivednon-adipocyte cells, which requires digesting adipose tissue withenzymes such as collagenase, and then separating the liberated cells bycentrifuging the digested sample. During centrifugation, theadipose-derived non-adipocyte cells separate from the adipocytes to forma pellet, whereas the lipid containing adipocytes float. Thenon-adipocyte cell fraction is then used as a source of tissue stemcells.

The present inventors have earlier described the use of an adiposetissue-derived cell suspension which comprises adipocytes for thepreparation of a pharmaceutical composition for use in the treatment ofan inflammatory disorder or the alleviation of pain associated with aninflammatory disorder in a subject, and for the treatment andalleviation of pain of conditions such as a cartilage or bone disorders.This is described in Australian Patent Application No. 2009201915 and inInternational Publication No. WO2010/020005, the contents of which areincorporated herein by cross-reference. The present inventors have alsoearlier described the use of adipose tissue-derived cells secretions forthe preparation of compositions for use in treatment of variousconditions and diseases, including in the alleviation of pain associatedwith such conditions.

Generations of selective breeding of animals for certain desirabletraits, such as rapid growth, efficient feed conversion and muscle massaccumulation in animals raised for meat production, or milk quality andvolume in dairy animals, has also resulted in modern breeds of animalsoften being prone to a higher incidence of detrimental health conditionsthan less intensively bred or selected animals, such as wildpopulations. The clinical incidence or effect of such detrimental traitscan be exacerbated by the manner in which the animals are farmed, suchas in intensive farming operations. Modern pig breeds, farmed underintensive conditions for example, are prone to leg weakness, such asosteochondrosis (OCD), arthritis, a high risk of clinical andsub-clinical bacterial infection, all of which have the potential todetrimentally affect the general well-being of the animal and hencedetrimentally affect the farming operation.

There remains a need for improved methods for the treatment ofinflammatory conditions, ligament and tendon injuries and compositionsfor use therein. There remains a need for improved methods for thetreatment and prevention of detrimental conditions associated withintensive animal farming. There remains a need for the treatment of painin a subject and compositions for use therein.

SUMMARY OF THE INVENTION

Methods previously described for the treatment of inflammatory disordersusing adipose tissue-derived cell suspensions and cell free compositionsteach the administration of the composition or suspension to theafflicted area, such as by intra-articular injection in the case of anarthritic joint. Surprisingly, the inventor has now identified thatdirect administration of the therapeutic composition to the afflictedarea is not required. The inventor has surprisingly identified that theremote delivery of a composition comprising secretions from an adiposetissue-derived cell suspension or of a composition comprising an adiposetissue-derived cell suspension, optionally comprising adipocytes, or ofa combination thereof, can also be effective in the treatment of suchconditions.

The inventors have surprisingly identified that frozen stem cells, suchas mesenchymal stem cells, such as adipose tissue-derived stem cells,may be used as therapeutic agents in treatment of various conditions.Surprisingly such frozen cells may be used without the need forculturing the cells after retrieval from frozen storage. The inventorshave also identified that storage of cells in the presence ofcell-derived secretions improves the viability and proliferationpotential of cryopreserved stem cells, including cells derived fromadipose tissue.

Accordingly, in a first aspect of the invention there is provided amethod of treating a condition selected from the group consisting of aninflammatory disorder, a ligament injury and a tendon injury, oralleviating pain associated with an inflammatory disorder, a ligamentinjury or a tendon injury, in a subject, comprising administering to thesubject a pharmaceutical composition which comprises (i) adiposetissue-derived cell secretions, or (ii) an adipose tissue-derived cellsuspension, optionally comprising adipocytes or (iii) a combination of(i) and (ii), wherein said administration to said subject is at a siteremote from the site of said condition. In an embodiment the subject isa mammalian subject. In an embodiment the adipose tissue-derived cellsuspension, optionally comprising adipocytes comprises aggregates ofcells and or comprises pieces of adipose tissue. In an embodiment thecell suspension comprises adipocytes. In an embodiment the cellsuspension is substantially free of adipocytes. In an embodiment theadipose tissue-derived cell secretions comprises a carrier liquidselected from cell culture media and distilled water. In an embodimentan adipose tissue-derived cell suspension, optionally comprisingadipocytes, or a combination of an adipose tissue-derived cellsecretions and an adipose tissue-derived cell suspension, optionallycomprising adipocytes comprises a carrier liquid being a cell culturemedium, such as DMEM.

In an embodiment the inflammatory disorder or condition is selected fromthe group consisting of osteoarthritis, stifle disease, wobblers, atendon injury and a ligament injury. In an embodiment the inflammatorydisorder is atopic dermatitis. In an embodiment the inflammatorydisorder or condition is selected from the group consisting ofrheumatoid arthritis, back pain, and multiple sclerosis. In anembodiment the inflammatory disorder or condition is an immune drivendisease. In an embodiment the method comprises administration of adiposetissue-derived cell secretions. In an embodiment the method comprisesadministration of an adipose tissue-derived cell suspension. In anembodiment the administration is subcutaneous administration. In anembodiment the administration is intra-muscular administration. In anembodiment the administration is in the rump, arm, or buttocks. In anembodiment the administration is into the neck of the subject, such asthe nape of the subject, such as the scruff of the neck when the subjectis a dog or cat.

In a second aspect there is provided a method of treating a jointdisease or condition in a subject, the method comprising administeringto the subject a pharmaceutical composition which comprises (i) adiposetissue-derived cell secretions, or (ii) an adipose tissue-derived cellsuspension, optionally comprising adipocytes, or (iii) a combination of(i) and (ii), wherein said administration to said subject is at a siteremote from the site of said condition. In an embodiment the subject isa mammalian subject. In an embodiment the adipose tissue-derived cellsuspension, optionally comprising adipocytes, comprises aggregates ofcells and or comprises pieces of adipose tissue. In an embodiment theadipose tissue-derived cell secretions comprises a carrier liquidselected from cell culture media and distilled water. In an embodimentan adipose tissue-derived cell suspension, optionally comprisingadipocytes, or a combination of an adipose tissue-derived cellsecretions and an adipose tissue-derived cell suspension, optionallycomprising adipocytes, comprises a carrier liquid being a cell culturemedium, such as DMEM. In an embodiment the treatment comprisesadministering a pharmaceutical composition comprising an adiposetissue-derived cell suspension to said mammal by subcutaneous injection.In an embodiment the subcutaneously administered cell suspension issubstantially free of adipocytes. In an embodiment the subcutaneouslyadministered cell suspension comprises adipocytes.

In an embodiment the mammalian subject is an equine, feline, canine,bovine or porcine animal. In an embodiment the subject is a human. In anembodiment the subject is poultry.

In an embodiment the administration is subcutaneous administration. Inan embodiment the administration is intra-muscular administration. In anembodiment the administration is in the rump, arm, or buttocks. In anembodiment the administration is into the neck of the subject, such asthe nape of the subject, such as the scruff of the neck when the subjectis a dog or cat.

In a third aspect of the invention there is provided a method for thetreatment or prevention of a disease in an intensively farmed animal,the method comprising administering to the animal a pharmaceuticalcomposition which comprises (i) adipose tissue-derived cell secretions,or (ii) an adipose tissue-derived cell suspension, optionally comprisingadipocytes, or (iii) a combination of adipose tissue-derived cellsecretions and an adipose tissue-derived cell suspension, optionallycomprising adipocytes, wherein said administration is by subcutaneousinjection or intramuscular injection.

In an embodiment the disease of an intensively farmed animal is anorthopeadic developmental disease. In an embodiment the disease of anintensively farmed animal is selected from the group consisting of legweakness, lameness, arthritis, developmental diseases and bacterialinfection. In an embodiment the developmental disease is osteochondrosis(OCD).

In an embodiment the pharmaceutical composition is administered to ananimal prior to the onset of clinical symptoms of the disease. In anembodiment the intensively fanned animal is a pig and the pharmaceuticalcomposition is administered prior to the onset of clinical symptoms ofthe orthopeadic developmental disease, such as osteochondrosis.

In an embodiment the intensively farmed animal is selected from thegroup consisting of pigs, cattle, sheep, and poultry,

In an embodiment the intensively farmed animal is a breeder female. Inan embodiment the intensively farmed animal is a pregnant female. In anembodiment the animal is a pregnant sow. In an embodiment the pregnantsow has clinical symptoms of osteochondrosis or arthritis.

In an embodiment the administration is subcutaneous administration. Inan embodiment the administration is intra-muscular administration. In anembodiment the administration is into the neck of the subject, such asthe nape of the subject.

The following embodiments apply to all aspects of the invention herein,unless the context clearly indicates otherwise.

In an embodiment the adipose tissue-derived cell secretions are preparedfrom an adipose tissue-derived cell suspension. In an embodiment theadipose tissue-derived cell suspension is substantially free ofadipocytes. In an embodiment the adipose tissue-derived cell suspensionfurther comprises adipocytes. In an embodiment the adiposetissue-derived cell suspension comprises mature adipocytes. In anembodiment the adipose tissue-derived cell secretions are prepared byculture of adipose tissue-derived cell suspension. In an embodiment theadipose tissue-derived cell suspension is substantially free ofadipocytes. In an embodiment the adipose tissue-derived cell suspensionfurther comprises adipocytes. In an embodiment the adiposetissue-derived cell suspension, optionally comprising adipocytescomprises aggregates of cells and or comprises pieces of adipose tissue.

In an embodiment the adipose tissue-derived cell suspension is preparedby a method that comprises removal of (i) part of the adipocyte contentor (ii) substantially all of the adipocyte content during preparation ofthe adipose tissue-derived cell suspension.

In an embodiment the adipose tissue-derived cell secretions is aconcentrated preparation. In an embodiment the concentrated preparationis concentrated in comparison to the cell secretions as initiallyharvested from the adipose tissue-derived cell suspensions or culturethereof. In an embodiment the adipose tissue-derived cell secretions isa preparation concentrated by between about 2-fold and about 20-fold. Inan embodiment the adipose tissue-derived cell secretions is apreparation concentrated by about 10-fold.

In an embodiment the adipose tissue-derived cell secretions is ofbovine, canine, porcine or equine origin. In an embodiment the adiposetissue-derived cell secretions is of human origin.

In an embodiment, the adipose tissue-derived cell secretions is derivedfrom adipose tissue autologous to the recipient subject or animal. In anembodiment the adipose tissue-derived cell secretions is derived fromadipose tissue allogeneic to the recipient subject or animal. In anembodiment the adipose tissue-derived cell secretions is derived fromadipose tissue xenogeneic to the recipient subject or animal.

In an embodiment the adipose tissue-derived cell suspension, optionallycomprising adipocytes, comprises mature adipocytes.

In an embodiment the adipose tissue-derived cell suspension, optionallycomprising adipocytes is of bovine, canine, porcine or equine origin. Inan embodiment the adipose tissue-derived cell suspension, optionallycomprising adipocytes is derived from adipose tissue autologous to therecipient subject or animal. In an embodiment the adipose tissue-derivedcell suspension, optionally comprising adipocytes is derived fromadipose tissue allogeneic to the recipient subject or animal. In anembodiment the adipose tissue-derived cell suspension, optionallycomprising adipocytes is derived from adipose tissue xenogeneic to therecipient subject or animal.

In an embodiment the adipose tissue-derived cell suspension, optionallycomprising adipocytes is a cell suspension obtained by cell expansion inculture.

In an embodiment the adipose tissue-derived cell secretions, or apharmaceutical composition thereof are stored frozen prior toadministration.

In an embodiment the adipose tissue-derived cell suspension, optionallycomprising adipocytes, or a pharmaceutical composition thereof arestored frozen prior to administration.

In an embodiment the adipose tissue-derived cell secretions incombination with adipose tissue-derived cell suspension, optionallycomprising adipocytes, or a pharmaceutical composition thereof is storedfrozen prior to administration. In an embodiment the cell secretions insaid combination are a concentrated preparation. In an embodiment thepreparation is concentrated by between 2-fold and 20-fold in comparisonto the secretions prior to concentration.

In an embodiment the method further comprises (i) thawing frozen adiposetissue-derived cell secretions, or (ii) thawing frozen adiposetissue-derived cell suspension, optionally comprising adipocytes, or(iii) thawing a frozen combination of adipose tissue-derived cellsecretions and an adipose tissue-derived cell suspension, optionallycomprising adipocytes, or (iv) thawing a frozen pharmaceuticalcomposition of any of (i), (ii) or (iii), prior to administration to therecipient subject or animal.

In an embodiment the frozen secretions, cell suspension, combinationthereof, or pharmaceutical composition thereof, is administered to therecipient subject or animal soon after thawing, such as within about 10minutes after thawing, or within about 20 minutes after thawing, orwithin about 30 minutes after thawing or within about one hour ofthawing or within about two hours of thawing.

In an embodiment the method further comprises combining (i) acomposition comprising adipose tissue-derived cell secretions and (ii)an adipose tissue-derived cell suspension, optionally comprisingadipocytes, prior to administering said combination to the recipientsubject or animal. In an embodiment said combining occurs within 2 hoursbefore said administration. In an embodiment one or both of saidcomposition comprising adipose tissue-derived cell secretions and saidadipose tissue-derived cell suspension, optionally comprisingadipocytes, is stored frozen prior to said combining. In a furtherembodiment the composition comprising adipose tissue-derived cellsecretions and said adipose tissue-derived cell suspension, optionallycomprising adipocytes, are mixed together before the composition isfrozen.

In an embodiment the pharmaceutical composition is a veterinarycomposition and the subject is a non-human animal.

In a fourth aspect of the invention there is provided use of (i) adiposetissue-derived cell secretions, or (ii) an adipose tissue-derived cellsuspension, optionally comprising adipocytes, or (iii) a combination of(i) and (ii), for the preparation of a pharmaceutical composition foruse in the treatment of a condition selected from the group consistingof an inflammatory disorder, a ligament injury and a tendon injury, oralleviating pain associated with an inflammatory disorder, a ligamentinjury or a tendon injury, in a mammalian subject, wherein thecomposition is suitable for administration to a site of said subjectremote from the site of said condition.

In a fifth aspect there is provided use of (i) adipose tissue-derivedcell secretions, or (ii) an adipose tissue-derived cell suspension,optionally comprising adipocytes, or (iii) a combination of (i) and(ii), for the preparation of a pharmaceutical composition for use in thetreatment of a joint disease or condition in a mammalian subject,wherein the composition is suitable for administration to a site of saidsubject remote from the site of said condition. In an embodiment theadipose tissue-derived cell suspension, optionally comprisingadipocytes, comprises aggregates of cells and or comprises pieces ofadipose tissue. In an embodiment the adipose tissue-derived cellsuspension is substantially free of adipocytes. In an embodiment theadipose tissue-derived cell suspension comprises adipocytes.

In a sixth aspect of the invention there is provided use of (i) adiposetissue-derived cell secretions, or (ii) an adipose tissue-derived cellsuspension, optionally comprising adipocytes, or (iii) a combination of(i) and (ii), for the preparation of a pharmaceutical composition foruse in the treatment or prevention of disease in an intensively farmedanimal, wherein the composition is suitable for subcutaneous injectionor intramuscular injection.

In a seventh aspect of the invention there is provided a compositioncomprising (i) adipose tissue-derived cell secretions, or (ii) anadipose tissue-derived cell suspension, optionally comprisingadipocytes, or (iii) a combination of (i) and (ii), for treatment of acondition selected from the group consisting of an inflammatorydisorder, a ligament injury and a tendon injury, or alleviating painassociated with an inflammatory disorder, a ligament injury or a tendoninjury, wherein the composition is administered to a site on a subjectremote from the site afflicted by the condition. In an embodiment thecomposition is an injectable composition. In an embodiment theadministration is by subcutaneous injection or intramuscular injection.

In an eighth aspect of the invention there is provided a compositioncomprising (i) adipose tissue-derived cell secretions, or (ii) anadipose tissue-derived cell suspension, optionally comprisingadipocytes, or (iii) a combination of (i) and (ii), for treatment of ajoint disease or condition in a mammalian subject, wherein saidcomposition is administered a site of said subject remote from the siteof said condition. In an embodiment the adipose tissue-derived cellsuspension, optionally comprising adipocytes, comprises aggregates ofcells and or comprises pieces of adipose tissue. In an embodiment theadipose tissue-derived cell suspension is substantially free ofadipocytes. In an embodiment the adipose tissue-derived cell suspensioncomprises adipocytes.

In a ninth aspect of the invention there is provided a compositioncomprising (i) adipose tissue-derived cell secretions, or (ii) anadipose tissue-derived cell suspension, optionally comprisingadipocytes, or (iii) a combination of (i) and (ii), for treatment orprevention of a disease in an intensively farmed animal, wherein in saidtreatment or prevention the composition is administered by subcutaneousinjection or intramuscular injection.

In a tenth aspect of the invention there is provided a pharmaceuticalcomposition comprising (i) adipose tissue-derived cell secretions, or(ii) an adipose tissue-derived cell suspension, optionally comprisingadipocytes, or (iii) a combination of (i) and (ii), together with apharmaceutically acceptable carrier, diluent, excipient or adjuvant. Inan embodiment the composition comprising adipose tissue-derived cellsecretions further comprises adipocytes. In an embodiment thecomposition is a frozen composition.

In an eleventh aspect of the invention there is provided a kitcomprising (a) a pharmaceutical composition selected from the groupconsisting of (i) a composition comprising adipose tissue-derivedsecretions, (ii) a composition comprising an adipose tissue-derived cellsuspension, optionally comprising adipocytes, and (iii) a combination of(i) and (ii); and (b) instructions for use of said kit in treatment of acondition selected from the group consisting of an inflammatorydisorder, a ligament injury and a tendon injury, or alleviating painassociated with an inflammatory disorder, a ligament injury or a tendoninjury; wherein said treatment comprises administration of saidpharmaceutical composition to a site on a subject remote from the siteafflicted by the condition.

In a twelfth aspect of the invention there is provided a kit comprising(a) a pharmaceutical composition selected from the group consisting of(i) a composition comprising adipose tissue-derived secretions, (ii) acomposition comprising an adipose tissue-derived cell suspension,optionally comprising adipocytes, and (iii) a combination of (i) and(ii); and (b) instructions for use of said kit in treatment of a jointdisease or condition in a mammalian subject, wherein said treatmentcomprises administration of said pharmaceutical composition to a site ona subject remote from the joint afflicted by the joint disease orcondition. In an embodiment the adipose tissue-derived cell suspension,optionally comprising adipocytes, comprises aggregates of cells and orcomprises pieces of adipose tissue. In an embodiment the adiposetissue-derived cell suspension is substantially free of adipocytes. Inan embodiment the adipose tissue-derived cell suspension comprisesadipocytes.

In a thirteenth aspect of the invention there is provided a kitcomprising (a) a pharmaceutical composition selected from the groupconsisting of (i) a composition comprising adipose tissue-derivedsecretions, (ii) a composition comprising an adipose tissue-derived cellsuspension, optionally comprising adipocytes, and (iii) a combination of(i) and (ii); and (b) instructions for use of said kit in treatment orprevention of disease in an intensively farmed animal, wherein in saidtreatment or prevention the composition is administered by subcutaneousinjection or intramuscular injection.

In an embodiment the kit comprises one or more frozen compositions. Inan embodiment the kit comprises instructions for combining a compositioncomprising adipose tissue-derived secretions and a compositioncomprising an adipose tissue-derived cell suspension, optionallycomprising adipocytes, prior to administration of a combinedcomposition. In an embodiment the kit further comprises one or moreinjection devices, such as one or more syringes. In an embodiment theinjection device contains a composition of the kit.

In a fourteenth aspect of the invention there is provided a method ofalleviating pain in a mammalian subject, the method comprisingadministering to the subject a pharmaceutical composition whichcomprises (i) adipose tissue-derived cell secretions, or (ii) an adiposetissue-derived cell suspension, optionally comprising adipocytes, or(iii) a combination of (i) and (ii), wherein said administration to saidsubject is by intramuscular injection or by subcutaneous injection or byan appropriate form of administration at or near a site of the pain. Inan embodiment the pain is associated with a condition selected from thegroup consisting of an inflammatory disorder, a ligament injury and atendon injury. In an embodiment the pain is associated withosteoarthritis, stifle disease, wobblers, a tendon injury or a ligamentinjury. In an embodiment the pain is associated with atopic dermatitis.In an embodiment the pain is associated with rheumatoid arthritis, backpain, or multiple sclerosis. In an embodiment the pain is associatedwith a condition selected from the group consisting of leg weakness,lameness, arthritis, developmental diseases and bacterial infection. Inan embodiment the pain is associated with osteochondrosis (OCD). In anembodiment the pain is associated with a burn injury. In an embodimentthe pain is neck and or shoulder pain, whiplash associated disorder, orcomplex regional pain syndrome. In an embodiment the pain is back pain.In an embodiment the pain is lower back pain. In an embodiment the painis pain associated with a sciatic disorder. In an embodiment thetreatment is of pain for which there is no discernable causativeclinical condition. In an embodiment the treatment is of pain for whichthere is no discernable causative clinical condition in the part orregion of the body in which the subject experiences the pain. In anembodiment the pain is neuropathic pain. In an embodiment theneuropathic pain is pain for which there is no discernable causativeclinical condition. In an embodiment the appropriate form ofadministration is injection. In an embodiment the appropriate form ofadministration is topical application. The neuropathic pain may belocalised to one area of the body or it may be experienced in multiplesites of the subject's body. When experienced in multiple sites, theintensity of the pain may be similar at multiple sites or it may bedifferent at multiple sites. In an embodiment the neuropathic pain isneuropathic facial pain. In an embodiment the pain is neuropathic facialpain and administration to said subject is by injection into the jaw orthe gum. In an embodiment the injection into the jaw or the gum is atthe original site of the pain. In an embodiment the pain is associatedwith a joint disease or joint disorder and the composition isadministered to a site on said subject which is remote to said joint. Inan embodiment the adipose tissue-derived cell suspension, optionallycomprising adipocytes, comprises aggregates of cells and or comprisespieces of adipose tissue.

In a fifteenth aspect of the invention there is provided use of (i)adipose tissue-derived cell secretions, or (ii) an adiposetissue-derived cell suspension, optionally comprising adipocytes, or(iii) a combination of (i) and (ii), for the preparation of apharmaceutical composition for use in alleviating pain in a mammaliansubject, wherein the pharmaceutical composition is suitable foradministration to said subject by intramuscular injection or bysubcutaneous injection or by an appropriate form of administration, suchas topical administration, at or near a site of the pain.

In a sixteenth aspect of the invention there is provided a compositioncomprising (i) adipose tissue-derived cell secretions, or (ii) anadipose tissue-derived cell suspension, optionally comprisingadipocytes, or (iii) a combination of (i) and (ii), for alleviating painin a mammalian subject, wherein the pharmaceutical composition isadministered to said subject by intramuscular injection or bysubcutaneous injection or by an appropriate form of administration, suchas topical administration, at or near a site of the pain.

In a seventeenth aspect of the invention there is provided a kitcomprising (a) a pharmaceutical composition selected from the groupconsisting of (i) a composition comprising adipose tissue-derivedsecretions, (ii) a composition comprising an adipose tissue-derived cellsuspension, optionally comprising adipocytes, and (iii) a combination of(i) and (ii); and (b) instructions for use of said kit in alleviatingpain in a mammalian subject, wherein the pharmaceutical composition isadministered to said subject by intramuscular injection or bysubcutaneous injection or by an appropriate form of administration, suchas topical administration, at or near a site of the pain.

In a further aspect the invention provides a composition comprisingadipose tissue-derived cells and adipose tissue-derived cell secretions.In an embodiment the cells are adherent cells. In an embodiment thecells are mesenchymal stem cells. In an embodiment the composition is afrozen composition. In an embodiment the adipose tissue-derived cellsecretions comprise clarified media from culture of adiposetissue-derived cells. In an embodiment the adipose tissue-derived cellsecretions is a concentrated preparation of media from culture ofadipose tissue-derived cells. In an embodiment the adiposetissue-derived cell secretions is a preparation concentrated between2-fold and 20-fold. In an embodiment the composition further comprisesadipocytes. In an embodiment the cells are progeny cells from culture ofan adipose tissue-derived cell suspension. In an embodiment the cellscomprise a cell line obtained by culture of an adipose tissue-derivedcell suspension. In an embodiment the progeny cells are from multiplepassaging of cells derived from an adipose tissue-derived cellsuspension. In an embodiment the multiple passaging comprises five ormore passages. In an embodiment the multiple passaging comprises ten ormore passages. In an embodiment the composition comprises cells of anadipose tissue-derived cell line which has been frozen multiple times.

In a further aspect the invention provides a method for thecryopreservation of a stored cell, the method comprising combining saidcell with a composition comprising cell secretions and storing saidcombination in a frozen state. In an embodiment the method is for thecryopreservation of a cell line. In an embodiment, prior to saidstoring, the combination is held at room temperature for up to one hour.In an embodiment the said stored cell is an adherent cell. In anembodiment the stored cell is a mesenchymal stem cell. In an embodimentthe stored cell is an adipose tissue-derived cell. In an embodiment thecell line is an adipose tissue-derived cell line. In an embodiment thecomposition comprising cell secretions comprises clarified media fromculture of an adipose tissue-derived cell suspension. In an embodimentthe culture of an adipose tissue-derived cell suspension is culture ofprogeny cells of an adipose tissue-derived cell suspension. In anembodiment the composition comprising cell secretions comprisesconcentrated media from culture of an adipose tissue-derived cellsuspension. In an embodiment the composition comprising cell secretionscomprises media from culture of an adipose tissue-derived cellsuspension concentrated between 2-fold and 20-fold. In an embodiment thecell line has been passaged multiple times. In an embodiment the cellline has been passaged more than five times. In an embodiment the cellline has been passaged more than ten times. In an embodiment the cellline has been passaged more than fifteen times. In an embodiment thecell line has been frozen multiple times.

It will be understood that embodiments described herein apply equally toany and all aspects of the invention described herein, they are simplynot repeated under each aspect of the invention for the sake of brevity.

The summary of the invention described above is not limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the preferred embodiments, as well asfrom the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Proliferation of cells after freezing. Cells that had not beenpassaged and were frozen without secretions (left graph) and withsecretions (right graph) were stained with the Click-iT ERD assay thatidentifies proliferating cells. The proliferating cells appear in theupper right quadrant.

FIG. 2: Recovery of cells after freezing. Cells that had been cultureduntil a cumulative cell doubling of approximately 13 times had beenreached were frozen without secretions (top image) and with secretions(bottom image) and then thawed and cultured for 72 hours.

FIG. 3: Paw volume measurements from a collagen antibody inducedarthritis (CAIA) mouse model treated intramuscularly with SVF+adipocytesecretions (▪) or vehicle control (●).

FIG. 4: Ankle size measurements from a collagen antibody inducedarthritis (CAIA) mouse model treated intramuscularly with SVF+adipocytesecretions (▪) or vehicle control (●).

FIG. 5: Clinical arthritis scores from a collagen antibody inducedarthritis (CAIA) mouse model treated intramuscularly with SVF+adipocytesecretions (▪) or vehicle control (●).

FIG. 6: Paw volume measurements from CAIA mice treated IV with cells orcells with concentrated secretions. Cells (▪); cells plus secretions(●).

FIG. 7: Paw volume Area Under the Curve results from CAIA mice treatedIV with cells or cells with concentrated secretions.

FIG. 8: Ankle size measurements from CAIA mice treated IV with cells orcells with concentrated secretions. Cells (▪); cells plus secretions(●).

FIG. 9: Ankle size Area Under the Curve results from CAIA mice treatedIV with cells or cells with concentrated secretions.

FIG. 10: Clinical arthritis scores from CAIA mice treated IV with cellsor cells with concentrated secretions. Cells (▪); cells plus secretions(●).

FIG. 11: Clinical arthritis score Area Under the Curve results from CAIAmice treated IV with cells or cells with concentrated secretions.

ABBREVIATIONS

DMEM Dulbecco's Modified Eagles Medium.

SVCs stromal vascular cells.

SVF stromal vascular fraction.

OCD osteochondrosis.

MSC mesenchymal stem cell(s).

Definitions

In the context of the present invention reference to a compositioncomprising “adipose tissue-derived secretions” will be understood tomean a composition which includes one or more factors released fromcells of the adipose tissue. The material used in the preparation of thecomposition comprising the secretions may or may not include adipocytes.

The term “pharmaceutically acceptable” as used herein in the context ofvarious components relevant to the invention, such as carriers,diluents, cryopreservatives, is intended to encompass not only suchcomponents which are suitable for administration to a human subject, butalso those suitable for administration to a non-human mammalian subject.In particular embodiments, the pharmaceutically acceptable component issuitable for administration to a non-human mammalian subject. Inparticular embodiments the pharmaceutically acceptable component issuitable for administration to a human subject. In particularembodiments, the pharmaceutically acceptable component is suitable foradministration to a non-human mammalian subject and to a human subject.

The terms “treating”, “treatment”, “therapy” and the like in the contextof the present specification refer to the alleviation of the symptomsand/or the underlying cause of the condition or disease, such asinflammatory disorder, ligament injury, or tendon injury or disease ofan intensively farmed animal. In certain embodiments a treatment willslow, delay or halt the progression of a disorder or the symptoms of thedisorder or injury, or reverse the progression of the disorder orinjury, at least temporarily. Hence, in the context of this inventionthe word “treatment” or derivations thereof such as “treating” when usedin relation to a therapeutic application includes all aspects of atherapy, such as the alleviation of pain associated with the conditionbeing treated, alleviation of the severity of the condition beingtreated, improvement in one or more symptoms of the condition beingtreated, etc. Use of the word “treatment” or derivatives thereof will beunderstood to mean that the subject being “treated” may experience anyone or more of the aforementioned benefits.

The term “preventing” and the like, in the context of the “prevention”of disease, refers to hindrance of the progression of the symptoms orthe underlying cause of the disease. It will be understood that completeprevention of a disease may occur, such that the disease does not occurin a treated animal or subject. Equally, it will be understood that theterm includes partial prevention, such as the failure of a disease toprogress to the typical state observed in an animal or subject leftuntreated.

Throughout this specification, reference to “a” or “one” element doesnot exclude the plural, unless context determines otherwise. Similarly,reference to “an embodiment” does not exclude the characteristic of thatdescribed embodiment applying in combination with one or more otherembodiments described, unless the context determines otherwise.

The term “therapeutically effective amount” as used herein includeswithin its meaning a non-toxic but sufficient amount of a compound orcomposition for use in the invention to provide the desired therapeuticeffect. The exact amount required will vary from subject to subjectdepending on factors such as the species being treated, the age andgeneral condition of the subject, co-morbidities, the severity of thecondition being treated, the particular agent being administered and themode of administration and so forth. Thus, for any given case, anappropriate “effective amount” may be determined by one of ordinaryskill in the art using only routine methods.

In the context of this specification, the term “comprising” meansincluding, but not necessarily solely including. Furthermore, variationsof the word “comprising”, such as “comprise” and “comprises”, havecorrespondingly varied meanings. Hence, the term “comprising” andvariations thereof is used in an inclusive rather than exclusive meaningsuch that additional integers or features may optionally be present in acomposition, method, etc. that is described as comprising integer A, orcomprising integer A and B, etc.

In the context of this specification the term “about” will be understoodas indicating the usual tolerances that a skilled addressee wouldassociate with the given value.

In the context of this specification, where a range is stated for aparameter it will be understood that the parameter includes all valueswithin the stated range, inclusive of the stated endpoints of the range.For example, a range of “5 to 10” will be understood to include thevalues 5, 6, 7, 8, 9, and 10 as well as any sub-range within the statedrange, such as to include the sub-range of 6 to 10, 7 to 10, 6 to 9, 7to 9, etc, and inclusive of any value and range between the integerswhich is reasonable in the context of the range stated, such as 5.5,6.5, 7.5, 5.5 to 8.5 and 6.5 to 9, etc.

In the context of this specification, the term “plurality” means anynumber greater than one.

It is to be noted that reference herein to use of the inventive methodsand compositions in treatment or therapy will be understood to beapplicable to human and non-human, such as veterinary, applications.Hence it will be understood that, except where otherwise indicated,reference to a patient, subject or individual means a human or anon-human, such as an individual of any species of social, economic,agricultural or research importance including but not limited to membersof the classifications of ovine, bovine, equine, porcine, feline,canine, primates, rodents, especially domesticated or farmed members ofthose classifications, such as sheep, cattle, horses, pigs and dogs.

Where examples of various embodiments or aspects of the invention aredescribed herein they will generally be prefaced by appropriate termsincluding “such as” or “for example”, or “including”. It will beunderstood that the examples are being described as inclusivepossibilities, such as for the purpose of illustration or understandingand are not, unless the context indicates otherwise, being provided aslimiting.

The pharmaceutical composition referred to herein may also be referredto as a medicament when intended for therapeutic use. Hence, it will beunderstood that where the invention is described as including the use ofa composition of described components for the preparation of apharmaceutical composition for an intended therapeutic purpose, thatdescription equally means use for the preparation of a medicament forthat intended therapeutic purpose, unless the context indicatesotherwise.

To the extent that it is permitted, all references cited herein areincorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have identified that surprisingly the remotedelivery of a composition comprising secretions from an adiposetissue-derived cell suspension can be effective in the treatment ofvarious conditions including inflammatory diseases and bone and jointdisorders, including ligament injuries and tendon injuries. Theinventors have also surprisingly identified that remote delivery of anadipose tissue-derived cell suspension, optionally comprisingadipocytes, or of a combination of adipose tissue-derived secretions andan adipose tissue-derived cell suspension, optionally comprisingadipocytes, can be effective in the treatment of such conditions.Previously described methods for the treatment of such conditions havedescribed the administration of the therapeutic to a site of disease orpain by direct application, such as intra-articular injection in thecase of a joint disease. The present invention thus pertains to methodsof treating such conditions by remote administration of (i) acomposition comprising adipose tissue-derived cell secretions, or (ii) acomposition comprising an adipose tissue-derived cell suspension,optionally comprising adipocytes, or (iii) a combination of (i) and(ii), to a subject in need thereof. The invention also provides for theuse of (i) adipose tissue-derived cell secretions, or (ii) an adiposetissue-derived cell suspension, optionally comprising adipocytes, or(iii) a combination of (i) and (ii), for the preparation of a medicamentfor the treatment of a condition selected from the group consisting ofan inflammatory disorder, a ligament injury and a tendon injury, oralleviating pain associated with an inflammatory disorder, a ligamentinjury, a tendon injury, neuropathic pain, or a burn injury, themedicament suitable for remote administration to a subject.

As described herein, the inventor has surprisingly identified thatadministration of such a therapeutic agent does not necessarily need tobe direct administration of the agent to the diseased or affected site,such as a joint. By administration of the therapeutic agent, such as bysubcutaneous injection or intramuscular injection, the inventor hasidentified that various diseases may be treated or prevented. In thecase of a disease affecting a joint, the description herein of theadministration as remote simply means that it is not administereddirectly into the joint, but rather is typically administered bysubcutaneous injection or intramuscular injection. Hence, the site ofadministration by subcutaneous injection or intramuscular injection mayor may not be particularly distant from the affected joint. Theinvention pertains also to methods for the treatment or prevention ofdisease in an intensively farmed animal, the method comprisingadministration by subcutaneous injection or intramuscular injection of(i) a composition comprising adipose tissue-derived cell secretions, or(ii) an adipose tissue-derived cell suspension, optionally comprisingadipocytes, or (iii) a combination of (i) and (ii).

The inventor has identified that allogeneic and xenogeneic compositionscan be used in the treatment and further that the therapeuticcompositions can be stored frozen prior to use. It will be understoodthat in an aspect the invention herein relates to such compositions perse, regardless of the manner in which they may be used or may beintended to be used. In other aspects, the invention relates to the useof compositions of the invention in the methods disclosed herein. Inthis manner so-called “off the shelf” or “ready to use” therapeuticproducts offering advantages, such as of supply, ease of use, lesspatient discomfort, and a lower requirement for technical skills,compared to an autologous patient-derived therapeutic agent, can be madeavailable. The present invention thus permits the preparation of thetherapeutic agent in advance of patient contact, such that a productcomprising adipose tissue-derived cell secretions or an adiposetissue-derived cell suspension, optionally comprising adipocytes, may bemade available without the need to anesthetize a subject or animal forextraction of adipose tissue. Similarly, where the therapeutic agent isa combination of adipose tissue-derived cell secretions and an adiposetissue-derived cell suspension, optionally comprising adipocytes, thatcombination may be made available to the user, such as a clinician,veterinarian, or farmer in advance or the separate compositions of cellsecretions and of cell suspension may be supplied, with the user thenpreparing the combination shortly before administration. As describedherein the cell secretions, the cell suspension, or the combination canbe stored, for example at −20° C. until required for use. Alternatively,the cell secretions, the cell suspension, or the combination may bestored at a lower temperature, such as in a freezer at −70° C. to −90°C., or in liquid nitrogen storage, either in the vapour phase or in theliquid phase, until required for use. Compositions comprising cells willtypically be stored in liquid nitrogen. In a preferred embodiment thecomposition comprising adipose tissue-derived cell secretions, or theadipose tissue-derived cell suspension, optionally comprisingadipocytes; or the combination of adipose tissue-derived cell secretionsand adipose tissue-derived cell suspension, optionally comprisingadipocytes, is stored in the liquid phase of liquid nitrogen storage. Inan embodiment the adipose tissue-derived cell secretions is aconcentrated preparation. In an embodiment the concentrated preparationis concentrated in comparison to the cell secretions as initiallyharvested from the adipose tissue-derived cell suspensions or culturethereof. In an embodiment the adipose tissue-derived cell secretions isa preparation concentrated by between about 2-fold and about 20-fold. Inan embodiment the adipose tissue-derived cell secretions is apreparation concentrated by about 10-fold.

Without wishing to be bound by any proposed mechanism of action, it isproposed that the adipose tissue-derived cell secretions comprisecytokines, such as anti-inflammatory cytokines, that are able to migrateto a source of injury or disease and there effect improvement in theunderlying condition, or alleviation of pain associated with thecondition. Similarly, the adipose tissue-derived cell suspension,optionally comprising adipocytes, when injected subcutaneously orintramuscularly then operate to secrete various cell factors, such ascytokines, which are able to migrate to the site of injury or disease,be it clinical or subclinical, thereby effecting improvement of theunderlying condition or prevention of clinical occurrence, for examplein an intensively farmed animal. As described in the Examples herein,the method is also effective in improving lameness of a treatedindividual and may provide benefits in performance enhancement, asdemonstrated by the improved agility and mobility of treatedindividuals.

Adipose Tissue

The cell secretions of the invention are adipose tissue-derived cellsecretions. The cell suspensions of the invention are adiposetissue-derived cell suspensions. Adipose tissue may be human adiposetissue or mammalian animal adipose tissue. The human or animal may bealive or dead, provided that there are still viable cells within theadipose tissue. The adipose tissue may comprise “white” adipose tissue,or “brown” adipose tissue.

The adipose tissue may originate from any source in the body which isaccessible. Subcutaneous fat, for example, is readily accessible withonly superficial wounding, or by using “keyhole surgery” techniques. Forexample adipose tissue may be tissue collected using liposuctiontechniques, or adipose tissue which is removed with reproductive tissuewhen de-sexing a male or female animal. The adipose tissue may be rinsedwith a tissue culture medium or buffered isotonic solution to removeadherent blood cells, and may be trimmed or coarsely processed to removelarge blood vessels or connective tissue elements prior to generating anadipose tissue-derived cell suspension.

The adipose tissue may be derived from a mature or from a juvenileanimal.

In particular embodiments the mammal is a companion animal, such as acanine or a feline domestic animal, or a working animal. In otherparticular embodiments the mammal is a, farm animal or racing animalselected from a horse, donkey, ass, cow, buffalo, sheep, goat, camel orpig.

Adipose Tissue-Derived Cell Suspension

The adipose tissue-derived cell secretions and hence the compositionscomprising such secretions, are preferably prepared by first obtainingor preparing an adipose tissue-derived cell suspension. As describedherein the methods, kits, uses, and compositions of the invention maycomprise adipose tissue-derived cell secretions, an adiposetissue-derived cell suspension or a combination of the cell secretionsand the cell suspension. The adipose tissue-derived cell suspension mayor may not comprise adipocytes.

The term “adipose tissue-derived cell suspension” as used hereinencompasses isolated cells from adipose tissue or small aggregates orpieces of adipose tissue, or a mixture of two or more of: isolatedcells, small aggregates and pieces of adipose tissue. The cellsuspension may be obtained by mechanically dissociating adipose tissueusing techniques which are readily available in the art. Any suitablemethod for the mechanical dissociation of adipose tissue may be used,for example by mincing adipose tissue with blades, or with scissors, orby forcing adipose tissue through screens or meshes with a pore sizesufficient to break the tissue into isolated cells or small pieces ofadipose tissue, or a combination of these techniques. Small aggregatesof adipose tissue may form when dissociated adipose-derived cellsreassociate into larger assemblies, for example on standing in a medium.Small pieces or aggregates of adipose tissue may be less than tenmillimeters in diameter, less than five millimeters in diameter, lessthan one millimeter in maximum diameter, less than 500 μm in maximumdiameter or less than 250 μm in maximum diameter.

The adipose tissue-derived cell suspension may be filtered through amesh or screen to remove cell aggregates or tissue pieces which aregreater than the mesh or screen pore size.

Proteolytic enzymes may be used to promote the dissociation of adiposetissue into an adipose tissue-derived cell suspension. Enzymes which aresuitable for such a use are well known in the art, and include but arenot limited to trypsin, and collagenase. It is usual to remove and/orotherwise inactivate the proteolytic enzymes before using theadipose-tissue-derived cell extract, as these enzymes may not becompatible with a desired in vivo use of the cells. The proteolyticenzymes may be used in combination with techniques for the mechanicaldissociation of adipose tissue to generate an adipose tissue-derivedcell suspension.

A mechanical dissociation technique may be used without using one ormore proteolytic enzymes. The technique used in this manner may be usedto rapidly generate an adipose tissue-derived cell suspension.

The cell suspension may be suspended in a liquid. The liquid may beadded to the adipose tissue before, during or after the dissociation ofthe adipose tissue. The liquid may comprise a medium which is capable ofmaintaining adipose tissue cell survival for at least 24 hours underappropriate culture conditions. The liquid may comprise an isotonicbuffered solution, such as a phosphate or a HEPES buffered saline, whichis capable of maintaining adipose tissue cell survival for at least onehour. The liquid may comprise a tissue culture medium. The liquid maycomprise serum or serum components which support or extend adiposetissue cell survival in the cell suspension. The serum or serumcomponents may be autologous serum or serum components.

In some embodiments the cell suspension may not have added liquid, butinstead the cells are suspended in liquid which is formed during thedissociation of the tissue.

The preparation of an adipose tissue-derived cell suspension maycomprise a centrifugation step. The centrifugation of isolated cells orsmall aggregates or pieces of adipose tissue suspended in a liquid, suchas a medium, is at approximately 500 g for 10 minutes, or for sufficienttime and at a sufficient g-force to generate a cell pellet whichcomprises adipose-derived non-adipocyte cells, above which is a layer ofmedium, floating above which in turn is a layer which comprises theviable adipocytes, and floating at the top is a layer of lipid which isderived from ruptured adipocytes. Following centrifugation, in certainembodiments the lipid layer and the medium layer will be discarded andthe retained cells are mixed, leaving an adipose tissue-derived cellsuspension which comprises viable adipocytes and adipose-derivednon-adipocyte cells. In other embodiments, only the layer comprising theviable adipocytes will be retained. In other embodiments, the layercomprising adipocytes may be removed and hence not included in theadipose tissue-derived cell suspension. This will typically occur whenpreparing an adipose tissue-derived cell suspension which issubstantially free of adipocytes. A cell suspension referred to hereinas being substantially free of adipocytes means that the cell suspensionhas been significantly depleted of adipocytes compared to the startingmaterial, such as by removal of the adipocyte fraction aftercentrifugation. It will be understood that substantially free ofadipocytes when used in relation to a cell suspension includes completeabsence of adipocytes and also includes the situation where minimalretention of adipocytes in the material has occurred. In otherembodiments, only part of the adipocyte content of the adipose tissuemay be removed in the preparation of the adipose tissue-derived cellsuspension. In this case, the resultant cell suspension will compriseadipocytes, but at a reduced proportion relative to other retainedcomponents, such as the stem cells, compared to the proportion in thestarting material. In an embodiment the adipose tissue-derived cellsuspension comprises at least 10% adipocytes by volume. In an embodimentthe adipose tissue-derived cell suspension comprises between 10% and 30%adipocytes by volume

One centrifugation step or multiple centrifugation steps may be used,for example to provide additional cell separation steps. In otherembodiments, the preparation of an adipose tissue-derived cellsuspension does not include a centrifugation step.

The adipose tissue-derived cell suspension may or may not compriseviable adipocytes. When present, the adipocytes may retain detectablequantities of lipid in their cytoplasm, and may be separated fromadipose-derived non-adipocyte cells on the basis of the differentdensity provided by the lipid. Lipid may be detectable using lightmicroscopy techniques, including phase contrast microscopy, or bystaining a sample of cells with a lipophilic dye such as Oil Red O.Adipocytes which retain lipid in their cytoplasm are considerably morefragile than other adipose-derived cells, and accordingly where viableadipocytes are desired techniques for dissociating tissue which damageor kill a large proportion of the adipocytes should be avoided. Theultrasonic dissociation of adipose tissue or techniques in which adiposetissue is vigorously shaken, for example, are unlikely to provide a cellsuspension which contains large numbers of viable adipocytes. Theviability of adipocytes may readily be determined using readilyavailable techniques, such as the LIVE/DEAD cell viability assays(Molecular Probes).

The adipose tissue-derived cell suspension may comprise both adipocytesand adipose-derived non-adipocyte cells. The adipose-derivednon-adipocyte sells typically include cells of the stromal vascularfraction, including mesenchymal stem cells. Cells of the stromalvascular fraction typically pellet upon centrifugation conditionsdescribed herein of an adipose tissue-derived cell suspension.

In embodiments which comprise both adipocytes and adipose-derivednon-adipocyte cells, the adipose tissue-derived cell suspension may beconveniently prepared by methods which comprise a centrifugation step,as described herein, in which both the adipocyte cell layer and thepelleted adipose-derived non-adipocyte cells are collected.Alternatively, in these embodiments the adipose tissue-derived cellsuspension may be prepared by dissociating adipose tissue as describedherein without a centrifugation step.

The adipose tissue-derived cell suspension, optionally comprisingadipocytes, may be stored under appropriate conditions. The storageconditions typically permit the retention of cell viability of some orall cells in the cell suspension, such as greater than 50%, greater than60%, greater than 70%, greater than 80%, greater than 90%, or greaterthan 95%.

Where the adipose tissue-derived cell suspension is to be stored frozenit may be in any carrier liquid appropriate for freezing of cells. As anillustrative but not limiting example, the cells may be suspended inculture medium, which may be serum-containing or serum-free, such asDMEM, RPMI, minimal essential media, or in serum prior to freezing.

Where the adipose tissue-derived cell suspension is to be stored frozenit typically also comprises a cryopreservative, for example,dimethylsulfoxide (DMSO) or glycerol, at an appropriate concentration,such as 5% to 10%. As described herein cell secretions such as thoseobtained or derived from cell culture of an adipose tissue-derived cellsuspension can also be used as a cryopreservative for cell storage. Suchsecretions, optionally clarified and or optionally concentrated, can becombined with a cell population intended for frozen storage, such asmesenchymal cells, such as adipose tissue-derived cells, including acell line resulting from culturing of an adipose tissue-derived cellsuspension. The combination may be held at an appropriate temperature,such as room temperature, for example approximately 18 C to 25 C, priorto freezing for any suitable time, such as up to about one or two hours,to permit interaction between the secretions and the cells, for exampleapproximately 10 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutesor two hours. In preferred embodiments the combination is held at roomtemperature for about 30 minutes.

The constituents of the cell suspension, such as the liquid medium andthe cryopreservative, are typically pharmaceutically acceptable at theconcentrations used. This has the advantage that the adiposetissue-derived cell suspension can be administered to a recipientsubject or animal after thawing with minimal post-thaw processing.

The cell suspension is typically frozen under controlled conditions tominimize cell damage, for example by slow freezing, typically at a rateof about 1° C./min, such as by placing in a programmable freezingdevice, or in an insulated container in a −70° C. to −90° C. freezer.For storage, frozen cells are typically then transferred to liquidnitrogen storage.

A cell processing method and device which may be used for thepreparation of adipose tissue-derived cell suspensions is described inco-pending application PCT/AU2012/000272, the contents of which areincorporated herein by reference.

Bovine Adipose Tissue Derived Cell Suspensions

The adipose tissue-derived cell suspensions, and hence the adiposetissue-derived cell secretions, may be derived from any appropriatesource. Bovine adipose tissue is one such source. The inventor haspreviously described inventive methods for the preparation of adiposetissue derived cell suspensions from bovine sources, particularly frombovine tail base tissue, as that material was found to be refractory tostandard methods appropriate to multiple other sources of adiposetissue, such as human, canine, equine, mouse and rat. This is describedin co-pending application PCT/AU2012/000274, the contents of which isincorporated herein by reference.

For example, where the present invention utilises bovine adipose-derivedmaterial, a bovine adipose tissue-derived cell suspension for use as atherapeutic composition or for use in the preparation of adiposetissue-derived cell secretions may be prepared according to a methodcomprising:

-   -   exposing a sample of bovine adipose tissue to a proteolytic        enzyme solution to generate a cell suspension;    -   centrifuging the suspension of cells to form a cell pellet, a        free lipid layer above a floating cell layer which comprises        adipocytes and an intermediate layer between the cell pellet and        the floating cell layer, said intermediate layer being to        depleted of cells relative to the cell pellet and the floating        cell layer; and    -   removing the free lipid layer and the intermediate layer and        mixing the cell pellet and floating cell layer to form an        adipose tissue derived cell suspension which comprises        adipocytes.

In the methods, uses and compositions of the invention where a cellsuspension which does not comprise adipocytes is desired, the floatingcell layer which comprises adipocytes may also be removed and discardedin the performance of the above method.

The method may comprise additional steps in the preparation of adiposetissue-derived cell suspensions as set out elsewhere in thisspecification, in particular the previous section headed “AdiposeTissue-Derived Cell Suspension”. These additional steps include, forexample, mechanically dissociating the tissue, and suspension via amedium or buffer etc.

The removed intermediate layer may be retained as it typically includesadipose tissue-derived secretions. As the concentration of secretions inthe removed intermediate layer is typically low compared to theconcentration of secretions produced from subsequent cell culturing ofadipose tissue-derived cell suspensions, as described below, cellsecretions harvested from cultured cells are typically used in themethods, uses and compositions of the invention.

In certain embodiments the proteolytic enzyme solution comprisescollagenase. The collagenase typically is used at a final concentrationof about 0.2% w/v or about 0.25% w/v or greater. In certain embodimentsthe exposure of the bovine adipose tissue to proteolytic enzyme isconducted under conditions which result in incomplete digestion of theadipose tissue, such as which result in significant amounts of intactadipose tissue being present. Typically, for example, there may bepieces of adipose tissue present that are the same size as they wereprior to starting the digestion. In embodiments of the method anywherebetween about 20% to about 80% of the adipose tissue may not bedigested.

In certain embodiments the cells may be subjected to multiplecentrifugation steps or wash steps, for example in order to removeexcessive free lipid.

As described further in the following section an adipose tissue-derivedcell suspension which may be of any species origin, such as mentionedherein, for example bovine, porcine, canine, feline, equine, human, etc,or an aliquot thereof, may be used in the preparation of a compositioncomprising secretions of the adipose tissue-derived cells.

Compositions Comprising Adipose Tissue-derived Secretions

A composition comprising secretions from adipose tissue-derived cellsmay be prepared from an adipose tissue-derived cell suspension by anyappropriate manner. As noted herein the liquid components formed duringthe preparation of an adipose tissue-derived cell suspension typicallyincludes adipose tissue-derived secretions, thereby representing oneembodiment of a composition comprising such secretions. In this form thecomposition comprising adipose tissue-derived secretions may becollected at any appropriate stage in the preparation of a cellsuspension, such as by collection of the intermediate liquid layerbetween the cell pellet and the floating cell layer after centrifugationof the adipose tissue-derived material. In this embodiment the collectedmaterial comprising secretions may or may not include adipocytes.

Typically, the composition is generated by exposure of a medium to theadipose tissue-derived cell suspension comprising adipocytes. Exposureof the medium to the adipocyte tissue-derived cell suspension may be forany appropriate time and conditions, as set by the operator. Exposure ofthe medium to the adipocyte tissue-derived cell suspension does notrequire conditions which enable cell attachment to a substratum. Inthese embodiments, the composition comprising adipose tissue-derivedsecretions may be generated by exposing a medium to the adiposetissue-derived cell suspension for any appropriate period of time, suchas at least 6 hours, at least 8 hours, at least 10 hours, or at least 12hours, followed by removal of the cell suspension from the medium, orvice versa, for example by centrifugation or by filtration. In anembodiment, the adipose tissue-derived cell suspension may be exposed tolow oxygen conditions, such as less than 10% oxygen, less than 5% oxygenor less than 1% oxygen. The removal of the cell suspension and themedium from each other may result in complete or incomplete removal ofcells. Hence the medium, which comprises the adipose tissue-derivedsecretions, may or may not include adipocytes after removal from thecell suspension. In certain embodiments the composition is generated byexposing a medium to the adipose tissue-derived cell suspension for nomore than 12 hours, no more than 18 hours or no more than 24 hours. Incertain embodiments the composition may be generated by exposing amedium to the adipose tissue-derived cell suspension for a longer periodof time, such as any period of time between about 1 and 15 days, forexample for about 1 day, 2 days, 3 days, 4, days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 15days. Typically, the suspension is kept in an incubator for 5 to 10 daysand then the secretions are collected. Exposure of the medium to thecells may or may not be under conditions which enable cell attachment toa substratum. Typically, where exposure of the medium to the cells isfor more than about 1 day the conditions will permit cell attachment tothe substratum.

The composition may comprise cell-derived molecules which are releasedfrom cells following cell death or the breakup of adipose tissue cells.The composition may comprise secretions of cells of the adiposetissue-derived cell suspension, optionally comprising adipocytes. Theexposing of a medium to an adipose tissue-derived cell suspension may beat a temperature of from 4° C. to 50° C., more typically at atemperature of from 10° C. to 40° C. and most typically at a temperatureof from 20° C. to 37° C.

For a typical adipose tissue-derived cell suspension, 5 g of adiposetissue is dissociated and suspended in 50 mls of DMEM containing 10%autologous serum. The adipose tissue derived cell suspension typicallycomprises from 100,000 to 1,000,000 non-adipocyte cells for every gramof adipose tissue source material. The number of adipocytes per gram ofadipose tissue source material is typically between 100,000 and5,000,000.

The term “medium” as used herein is intended to encompass compositionswhich support the survival of at least some cells in an adiposetissue-derived cell suspension for at least one hour. The medium may bea tissue culture medium, such as DMEM, RPMI, or minimal essentialmedium, optionally supplemented with serum. The medium may be a bufferedisotonic solution, such as a phosphate buffered saline or Hank'sbuffered saline solution, provided the medium is suitable foradministration to a subject. The medium may be liquid which is formedduring the dissociation of adipose tissue. The medium may optionally besupplemented with factors which promote cell survival or attachment andcell division, such as insulin, progesterone and selenium, or serum orserum components. In certain embodiments the medium must be suitable fora pharmaceutical composition, which is acceptable for in vivo use. Suchmedia will be substantially free of pyrogens or other impurities whichmay be harmful to humans or animals. Pharmaceutically-acceptable mediaare commercially available. The phrase “pharmaceutically acceptable”refers to molecular entities and compositions that do not produceunacceptable adverse, allergic, or other untoward reactions whenadministered to an animal or a human.

Preparation of a composition comprising adipose tissue-derivedsecretions may include a step of lysis of the adipose tissue-derivedcell suspension comprising adipocytes. A lysate comprising cellsecretions may be prepared by any suitable method. In an exampleembodiment, an adipose tissue-derived cell suspension may be exposed toa medium, such as described above. Cells of the suspension may then belysed by any suitable means, such as by mechanical disruption (eg,vigorous shaking or agitation), ultrasonic disruption, freeze thawing,freeze drying or the addition of one or more agents capable of inducingcell lysis, typically adipocyte lysis. Such lysing agents are known inthe art and include urea, sodium dodecyl sulphate and Triton ×100. Aftera lysis step the preparation may be centrifuged or filtered to assist inthe removal of cell debris, or it may be used without such a clarifyingstep, in which case the composition comprising adipose tissue-derivedsecretions may also include cell debris. In some cases the cell lysatemay be removed from the lysing agent by precipitation of the celllysate. Where the lysis step results in incomplete cell lysis, thecomposition comprising adipose tissue-derived secretions may alsocomprise adipose-derived cells, such as adipocytes.

In certain embodiments preparation of the adipose tissue-derivedsecretions comprises culturing the cell suspension comprising adipocytesunder appropriate conditions to form an adherent cell culture, such as aconfluent adherent cell culture; harvesting supernatant of the adherentcell culture and removing cells from said supernatant to form acomposition comprising adipose tissue-derived secretions. The removal ofthe cells from the supernatant to leave a composition comprising adiposetissue-derived secretions may be complete removal or may be partialremoval. In the latter case, the composition comprising adiposetissue-derived secretions may therefore also include adipocytes.

Prior to commencing the culturing of the cells, the adipose tissuederived cell suspension may be re-suspended in a desired volume of anappropriate buffer, such as DMEM, RPMI or minimal essential media. Thecell suspension, or an aliquot thereof, may be added to a sterile tissueculture flask and incubated under appropriate conditions, typicallyuntil the adherent cells have reached confluence. The cell culture ispreferably in the presence of sterile serum. The concentration of theserum in the culture may be any suitable concentration which assistsculturing of adipose tissue-derived cells, such as for example in therange of about 5% v/v to about 30% v/v, such as about 10% v/v, or about15% v/v or about 20% v/v. The serum may be any appropriate serum for theculturing of adipose tissue-derived cells, such as a commercial fetalcalf serum, or a serum prepared in house, such as by methods known inthe art. The serum may be autologous, having been prepared from the sameindividual from which the adipose tissue was obtained, or it may beallogeneic. In other embodiments the serum may be xenogeneic, such asthe use of calf serum in the culturing of adipose tissue-derived cellsobtained from a canine source. Typically, the cells are cultured at 37°C. with 5% CO₂. In a further embodiment the cells are cultured inhypoxic conditions.

During culturing the adipose tissue-derived cells secrete cytokinesincluding anti-inflammatory molecules, pro-inflammatory molecules,chemokines, growth factors and other cell signalling molecules into themedium. The supernatant in the culture thereby comprises adiposetissue-derived secretions.

In certain embodiments the culture may be frozen and freeze dried,resulting in a freeze dried preparation that includes cells and thesecretions. Rehydration of the freeze dried preparation will lyse themajority of the cells resulting in the release of additional cytokines.Rehydration will typically be performed using a volume of fluid that isless than the original volume of the adipose tissue-derived cells, suchas a volume of fluid that is between about 5 and about 20 times lessthan the original volume, more typically about 10 times less than theoriginal volume of the adipose tissue-derived cells which results in acomposition that is 10 times concentrated. The composition may then befiltered to remove cell debris resulting in a composition that containsconcentrated cytokines. This provides a preferred method for producinglarge volumes of concentrated secretions.

In other certain embodiments the supernatant may be harvested from theculture at any appropriate time, although typically for an adherent cellculture it will be harvested when the cells have reached confluence,such as after about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days.Cells, cell debris and any remaining adipose tissue may be removed fromthe supernatant, such as by filtration. In an embodiment the filtrationmay be through a 20 micron mesh. If desired, multiple steps offiltration may be undertaken such as through two or more filters ofdecreasing mesh size. The resultant preparation of adiposetissue-derived secretions is typically filter sterilised, such asthrough a 0.22 micron filter. The sterilised composition may be usedimmediately, or may be aliquoted for use, or for storage. Typically, ifstored, the composition is stored frozen at −20° C. The compositioncontains secretions from the adipose tissue-derived cells.

A composition comprising adipose tissue-derived secretions may alsocomprise adipocytes. Where present, the adipocytes may remain from theoriginal adipose tissue used in the preparation of the secretions orthey may be added to the composition comprising the secretions.

As described above the preparation of adipose tissue-derived cellsecretions may be made by culturing an adipose tissue-derived cellsuspension and subsequent harvest and filtration of supernatant, with orwithout additional steps described above, such as filter-sterilizing,freeze-drying and concentrating.

A composition comprising the secretions, which may be a harvestedsupernatant comprising secretions, may, for example, be freeze-dried andsubsequently re-hydrated in a desired volume of an appropriate liquid.Typically the appropriate liquid will be pharmaceutically acceptable.The appropriate liquid may be distilled water.

Optionally, confluent adipose tissue-derived cell cultures, afterharvest of supernatant, may be passaged into new tissue culture flasksand cultured again to confluence under appropriate conditions. Continuedpassaging of the cells may be undertaken for the preparation of adiposetissue-derived secretions. For example, supernatant may be harvestedfrom flasks in any desired passage number, for example from flasks inpassage number three through to passage five. Harvested supernatants maybe pooled as desired, for example to obtain a greater amount ofsecretions. With additional steps, such as concentration steps, in thismanner an adipose tissue-derived cell secretion preparation of higherconcentration may be prepared.

Pharmaceutical Compositions and Other Compositions of the Invention

In aspects of the invention the adipose tissue-derived composition,containing secretions of adipose tissue cells, or the adiposetissue-derived cell suspension, optionally comprising adipocytes, isused for the preparation of a pharmaceutical composition. According toone aspect the invention provides (i) a composition comprising adiposetissue-derived secretions or (ii) an adipose tissue-derived cellsuspension, optionally comprising adipocytes or (iii) a combination of(i) and (ii) for the preparation of a pharmaceutical composition for usein the treatment of a condition selected from the group consisting of aninflammatory disorder, a ligament injury and a tendon injury in asubject, or for use in alleviating pain associated with an inflammatorydisorder, a ligament injury; a tendon injury, neuropathic pain, or aburn injury, wherein the treatment comprises remote administration ofthe pharmaceutical composition to the subject. The pharmaceuticalcomposition may also be referred to as a medicament.

Typically, the remote administration is subcutaneous administration orintra-muscular administration. Typically the pharmaceutical compositionalso comprises one or more of a pharmaceutically acceptable carrierdiluent, excipient or adjuvant.

In another aspect the invention provides use of (i) a compositioncomprising adipose tissue-derived secretions or (ii) an adiposetissue-derived cell suspension, optionally comprising adipocytes, or(iii) a combination of (i) and (ii), for the preparation of apharmaceutical composition for use in the treatment or prevention ofdisease in an intensively farmed animal, wherein the pharmaceuticalcomposition is suitable for subcutaneous injection or intramuscularinjection.

In another aspect the invention provides use of use of (i) a compositioncomprising adipose tissue-derived cell secretions, or (ii) an adiposetissue-derived cell suspension, optionally comprising adipocytes, or(iii) a combination of (i) and (ii), for the preparation of apharmaceutical composition for use in the treatment of a joint diseaseor condition in a mammalian subject, wherein the composition is suitablefor administration to a site of said subject remote from the site ofsaid condition.

In another aspect the invention provides use of (i) adiposetissue-derived cell secretions, or (ii) an adipose tissue-derived cellsuspension, optionally comprising adipocytes, or (iii) a combination of(i) and (ii), for the preparation of a pharmaceutical composition foruse in alleviating pain in a mammalian subject, wherein thepharmaceutical composition is suitable for administration to saidsubject by intramuscular injection or by subcutaneous injection or by anappropriate form of administration, such as by topical administration,at or near a site of the pain.

The invention thus provides pharmaceutical compositions or medicamentscomprising (i) adipose tissue-derived cell secretions, or (ii) anadipose tissue-derived cell suspension, optionally comprisingadipocytes, or (iii) a combination of (i) and (ii). The pharmaceuticalcomposition may be provided as part of a kit, for example includingadditional components useful in the intended treatment, such as forexample written instructions, or it may be provided as a single item,such as a single vial or aliquot of the composition.

According to a further aspect the invention provides a pharmaceuticalcomposition comprising adipose tissue-derived secretions, together witha pharmaceutically acceptable carrier, diluent, excipient or adjuvant.In certain embodiments the composition comprising adipose tissue-derivedsecretions further comprises adipocytes. In certain embodiments thecomposition is cell-free. In certain embodiments the composition isprepared by exposing a medium or other liquid to an adiposetissue-derived cell suspension, which may or may not comprise matureadipocytes. The pharmaceutical composition has therapeutic properties,such as for the treatment of a condition selected from the groupconsisting of an inflammatory disorder, a ligament injury and a tendoninjury in a subject, or for alleviating pain associated with a conditionselected from the group consisting of an inflammatory disorder, aligament injury, a tendon injury, neuropathic pain and a burn injury.The pharmaceutical composition has therapeutic properties suitable fortreatment or prevention of disease in an intensively farmed animal. Suchdiseases include leg weakness, lameness, arthritis, developmentaldiseases and bacterial infection. In an embodiment the developmentaldisease is osteochondrosis (OCD). In an embodiment the developmentaldisease is a bone cyst. In an embodiment the developmental disease is anorthopaedic developmental disease.

In embodiments, the adipose tissue is taken from an individual subject,and the pharmaceutical composition is administered to the sameindividual, and thus the adipose tissue-derived secretions or adiposetissue-derived cell suspension, optionally comprising adipocytes, orcombination thereof, is a purely autologous preparation.

In embodiments, the adipose tissue is taken from one or more individualsubjects and the pharmaceutical composition is administered to adifferent subject of the same species, and thus the adiposetissue-derived secretions or adipose tissue-derived cell suspension,optionally comprising adipocytes, or combination thereof is anallogeneic preparation. In embodiments the adipose tissue is taken froman individual of a different species to that which is intended to be arecipient of the therapeutic composition. For example, a compositioncomprising secretions or cells or a combination of both prepared frombovine tissue may be for administration to an individual of a differentspecies, such as a human, feline, porcine, or canine. In embodimentswhere the composition comprising adipose tissue-derived secretions oradipose tissue-derived cell suspension, optionally comprisingadipocytes, is for use in a different individual of the same species asthe source material or for use in an individual of a different speciesas the source material, the composition may typically be devoid of cellsof the immune system in order to minimise the possibility of host(recipient) immune response to the composition or graft versus hostdisease.

In embodiments the pharmaceutical composition is prepared from more thanone source of adipose tissue, such as from different preparations takenfrom the same individual or from different preparations taken fromdifferent individuals. The pooling may comprise combining multipleadipose tissue-derived cell suspensions, such as in a pooled culturingstep or the pooling may comprise combining multiple compositions ofadipose tissue-derived secretions, such as may be obtained from separateculturing or exposure steps.

The pharmaceutical composition may be administered to the subjectpatient at a site remote from the afflicted area. In this context,“remote” means that the administration is not direct application of thecell secretions or cell suspension or combination thereof to the site ofinflammation or other injury or disease being treated where such a siteis identifiable. As an illustration, in the case of treatment of anarthritic joint, administration as previously described in the artinvolved injection of adipose tissue-derived cell suspensions or adiposetissue-derived cell secretions directly into the afflicted joint. Suchadministration requires a high degree of skill on the part of thetreating physician or clinician to ensure appropriate precision. Thehandling of the affected limb or joint required in such administrationalso increases the distress experienced by the patient, be they human ornon-human. By providing for the remote administration of adiposetissue-derived cell secretions, or adipose tissue-derived cellsuspension, or combination thereof, the present invention offersimproved methods, uses and compositions for the treatment of suchdiseases. For example, the remote administration may be by subcutaneousinjection, such as in the scruff of the neck of an animal (for example acat or dog) being treated, or by intramuscular injection. As a furtherexample, administration to a dog by intramuscular injection may be in tothigh of the dog. As a further example, administration to a bovine byintramuscular injection may be in the caudal fold.

Where the therapeutic compositions of the invention are administered totreat or prevent a disease of an intensively farmed animal, theadministration is by subcutaneous injection or intramuscular injection.Due to the nature of the condition being treated in such an animaldescription of the administration being “remote” may not be appropriate.For example, administration of the therapeutic composition of theinvention to an intensively farmed animal may be for treatment orprevention of generalized or localized leg weakness, osteochondrosis. Asdescribed herein administration of the therapeutic composition to suchanimals is by subcutaneous injection or intramuscular injection.

Where the therapeutic compositions of the invention are administered fortreatment of pain for which there is no discernable clinical cause, orfor neuropathic pain which may or may not have a discernable clinicalcause, the composition may be administered to the subject by anyappropriate means, such as by injection or by topical application, whichmeans may be at or near a site of the pain or may be remote from a siteof the pain. Neuropathic pain may be experienced by a subject atmultiple sites in the body. Treatment of such pain may be byadministration at one site, such as a site of original pain, which sitemay also be remote from another site of pain experienced by the subject.In an example, the pain is neuropathic pain. The neuropathic pain may belocalised to one area of the body or it may be experienced in multiplesites of the subject's body. When experienced in multiple sites, theintensity of the pain may be similar at multiple sites or it may bedifferent at multiple sites. An example of neuropathic pain isneuropathic facial pain. In methods of the invention, neuropathic facialpain in a subject may be treated by administration of a composition ofthe invention to the subject by injection into the jaw or the gum. Theinjection into the jaw or the gum of such a patient may compriseadministration at the original site of the pain or may be administrationat a secondary site of the pain.

As shown in the Examples compositions and methods of the invention arealso effective in alleviating pain and blistering associated with burns.Where the compositions of the invention are administered for treatmentof a burn injury, or for alleviating pain associated with a burn of theskin, the composition is typically administered by topical application,such as in the form of a cream, gel, or lotion.

A pharmaceutical composition of the invention may be supplied to theuser as a frozen solution. As described herein, for example, the cellsecretions, the cell suspension, or the combination can be stored, atapproximately −20° C. until required for use. Alternatively, the cellsecretions, the cell suspension, or the combination may be stored at alower temperature, for example in a freezer at −70° C. to −90° C., or inliquid nitrogen storage, either in the vapour phase or in the liquidphase, until required for use. Compositions comprising cells willtypically be stored in liquid nitrogen. In a preferred embodiment thecomposition comprising adipose tissue-derived cell secretions, or theadipose tissue-derived cell suspension, optionally comprisingadipocytes, or the combination of adipose tissue-derived cell secretionsand adipose tissue-derived cell suspension, optionally comprisingadipocytes, is stored in the liquid phase of liquid nitrogen storage. Ina preferred embodiment a composition comprising cells is stored incombination with cell secretions obtained from a culture of adiposetissue-derived cells. In another preferred embodiment a compositioncomprising cells is stored in a composition which comprises clarifiedand or concentrated media from a culture of adipose tissue-derivedcells. The culture of adipose tissue-derived cells will be understood toinclude culture of cells freshly obtained from adipose tissue, as wellas culture of cells which have arisen as progeny cells or a cell linefrom prior culturing. In use, such as by the treating physician,clinician, veterinarian, technician, assistant, or farmer, thecomposition is typically administered to the subject or animal as soonas possible after thawing. The pharmaceutical composition mayalternatively be stored, for example on ice or in a refrigerator or in acool pack, at approximately 2° C. to 5° C. for a short time betweenthawing and administration. In this context a short time would typicallybe no more than several hours, such as no more than about half an hour,or no more than about one hour, or no more than about two hours. As thecryoprotectants are typically toxic to the cells and can cause loss ofviability if kept thawed, the composition, particularly where itcomprises viable cells, is typically injected to the recipient animal assoon as possible after thawing.

It had previously been considered that a stored (eg., a frozen)suspension comprising mesenchymal stem cells requires a period of timeunder appropriate cell culturing conditions before use in a therapeuticsetting, for example to assist in cell recovery. The inventor hassurprisingly identified that culturing of a previously frozen cellsuspension, such as an adipose tissue-derived cells suspensionoptionally comprising adipocytes, is not required prior to use of thecell suspension in a therapeutic setting. As described herein benefitsfrom the administration of such a cell suspension are apparent when thecell suspension is administered after thawing and without interveningcell culturing. As demonstrated in the Examples herein, storage of cellsin the presence of adipose tissue-derived cell secretions providesfurther benefits. For example, frozen cells stored in the presence ofsecretions, optionally a clarified and or concentrated preparation ofsecretions, demonstrate superior therapeutic efficacy when utilisedafter retrieval from storage compared to frozen cells stored in theabsence of secretions.

A pharmaceutical composition of the invention may be supplied in a“ready-to-use” form. In such embodiments the user typically requiresonly thawing to an acceptable temperature for administration before thecomposition is administered. In such embodiments the composition may besupplied in pre-measured doses, such as a pre-measured or pre-determineddose suitable for a given recipient subject or animal, for examplepre-determined on the basis of the recipient species, or on the basis ofthe recipient individual, such as a small breed of dog, compared to alarge breed of dog, or a juvenile animal compared to an adult animal.The pre-measured dose may alternatively or additionally be on the basisof the disease or condition being treated or intended to be prevented. Aready-to-use form of the composition may comprise the compositionsupplied with or in an injectable device, such as a syringe. Theinjectable device may be capable of delivering a single application toan individual recipient or may be capable of delivering single ormultiple applications to multiple recipients. The injectable device maybe adjustable, for example to permit delivery of a range of differentdoses.

In embodiments where the pharmaceutical composition comprises acombination of adipose tissue-derived cell secretions and an adiposetissue-derived cell suspension, optionally comprising adipocytes, thecomposition may be supplied to the user as a combination or as separatecompositions for combination by the user. It will be understood thatreference to a “user” in this context means the individual who actuallyadministers the therapeutic composition to the recipient subject oranimal and also means a member of the team or group who is undertakingthat administration. For example the user may be any individual who isassisting in the application of the methods of the invention such as aclinician, a doctor, a veterinarian, a farmer, a clinical nurse, aveterinary nurse, a technical assistant, or a farmhand.

Inflammatory Disorders

The pharmaceutical composition may be administered for the treatment ofan inflammatory disorder and/or for alleviating pain associated with aninflammatory disorder in a subject.

Inflammation may arise as a response to an injury or abnormalstimulation caused by a physical, chemical, or biologic agent. Aninflammation reaction may include the local reactions and resultingmorphologic changes, destruction or removal of the injurious material,and responses that lead to repair and healing. The term “inflammatory”when used in reference to a disorder refers to a pathological processwhich is caused by, resulting from, or resulting in inflammation that isinappropriate or which does not resolve in the normal manner.Inflammatory disorders may be systemic or localized to particulartissues or organs.

Inflammation is known to occur in many disorders which include, but arenot limited to: Systemic Inflammatory Response (SIRS); Alzheimer'sDisease (and associated conditions and symptoms including: chronicneuroinflammation, glial activation; increased microglia; neuriticplaque formation; and response to therapy); Amyotropic Lateral Sclerosis(ALS), arthritis (and associated conditions and symptoms including, butnot limited to: acute joint inflammation, antigen-induced arthritis,arthritis associated with chronic lymphocytic thyroiditis,collagen-induced arthritis, juvenile arthritis; rheumatoid arthritis,osteoarthritis, prognosis and streptococcus-induced arthritis,spondyloarthopathies, gouty arthritis), asthma (and associatedconditions and symptoms, including: bronchial asthma; chronicobstructive airway disease; chronic obstructive pulmonary disease,juvenile asthma and occupational asthma); cardiovascular diseases (andassociated conditions and symptoms, including atherosclerosis;autoimmune myocarditis, chronic cardiac hypoxia, congestive heartfailure, coronary artery disease, cardiomyopathy and cardiac celldysfunction, including: aortic smooth muscle cell activation; cardiaccell apoptosis; and immunomodulation of cardiac cell function; diabetesand associated conditions, including autoimmune diabetes,insulin-dependent (Type 1) diabetes, diabetic periodontitis, diabeticretinopathy, and diabetic nephropathy); gastrointestinal inflammations(and related conditions and symptoms, including celiac disease,associated osteopenia, chronic colitis, Crohn's disease, inflammatorybowel disease and ulcerative colitis); gastric ulcers; hepaticinflammations such as viral and other types of hepatitis, cholesterolgallstones and hepatic fibrosis, HIV infection and associatedconditions, including degenerative responses, neurodegenerativeresponses, and HIV associated Hodgkin's Disease, Kawasaki's Syndrome andassociated diseases and conditions, including mucocutaneous lymph nodesyndrome, cervical lymphadenopathy, coronary artery lesions, edema,fever, increased leukocytes, mild anemia, skin peeling, rash,conjunctiva redness, thrombocytosis; inflammatory disorders of the skin,including dermatitis, such as atopic dermatitis and associatedconditions; multiple sclerosis, nephropathies and associated diseasesand conditions, including diabetic nephropathy, endstage renal disease,acute and chronic glomerulonephritis, acute and chronic interstitialnephritis, lupus nephritis, Goodpasture's syndrome, hemodialysissurvival and renal ischemic reperfusion injury, neurodegenerativediseases and associated diseases and conditions, including acuteneurodegeneration, induction of IL-I in aging and neurodegenerativedisease, IL-I induced plasticity of hypothalamic neurons and chronicstress hyperresponsiveness, ophthalmopathies and associated diseases andconditions, including diabetic retinopathy, Graves' ophthalmopathy, anduveitis, osteoporosis and associated diseases and conditions, includingalveolar, femoral, radial, vertebral or wrist bone loss or fractureincidence, postmenopausal bone loss, mass, fracture incidence or rate ofbone loss, otitis media (adult or paediatric), pancreatitis orpancreatic acinitis, periodontal disease and associated diseases andconditions, including adult, early onset and diabetic; pulmonarydiseases, including chronic lung disease, chronic sinusitis, hyalinemembrane disease, hypoxia and pulmonary disease in SIDS; restenosis ofcoronary or other vascular grafts; rheumatism including rheumatoidarthritis, rheumatic Aschoff bodies, rheumatic diseases and rheumaticmyocarditis; thyroiditis including chronic lymphocytic thyroiditis;urinary tract infections including chronic prostatitis, chronic pelvicpain syndrome and urolithiasis, immunological disorders, includingautoimmune diseases, such as alopecia aerata, autoimmune myocarditis,Graves' disease, Graves ophthalmopathy, lichen sclerosis, multiplesclerosis, psoriasis, systemic lupus erythematosus, systemic sclerosis,thyroid diseases (e.g. goitre and struma lymphomatosa (Hashimoto'sthyroiditis, lymphadenoid goitre), sleep disorders and chronic fatiguesyndrome and obesity (non-diabetic or associated with diabetes),resistance to infectious diseases, such as Leishmaniasis, Leprosy, LymeDisease, Lyme Carditis, malaria, cerebral malaria, meningitis,tubulointerstitial nephritis associated with malaria), which are causedby bacteria, viruses (e.g. cytomegalovirus, encephalitis, Epstein-BarrVirus, Human Immunodeficiency Virus, Influenza Virus) or protozoans(e.g., Plasmodium falciparum, trypanosomes), response to trauma,including cerebral trauma (including strokes and ischemias,encephalitis, encephalopathies, epilepsy, perinatal brain injury,prolonged febrile seizures, SIDS and subarachnoid hemorrhage), low birthweight (e.g. cerebral palsy), lung injury (acute hemorrhagic lunginjury, Goodpasture's syndrome, acute ischemic reperfusion), myocardialdysfunction, caused by occupational and environmental pollutants (e.g.susceptibility to toxic oil syndrome silicosis), radiation trauma, andefficiency of wound healing responses (e.g. burn or thermal wounds,chronic wounds, surgical wounds and spinal cord injuries), septicemia,hypothyroidism, oxygen dependence, cranial abnormality, early onsetmenopause, a subject's response to transplant (rejection or acceptance),acute phase response (e.g. febrile response), general inflammatoryresponse, acute respiratory distress response, acute systemicinflammatory response, wound healing, adhesion, immunoinflammatoryresponse, neuroendocrine response, fever development and resistance,acute-phase response, stress response, disease susceptibility,repetitive motion stress, tennis elbow, ligament and tendon problems,and pain management and response.

In particular embodiments the inflammatory disorder is a joint-relatedinflammatory disorder, such as arthritis.

The methods and compositions of the invention may be used for thetreatment of ligament injuries and tendon injuries or for thealleviation of pain associated with such injuries. Ligament injuries andtendon injuries, in some forms, can be classified as inflammatorydisorders. Some ligament injuries and tendon injuries may not beconsidered inflammatory disorders. For the avoidance of doubt, ligamentinjuries and tendon injuries contemplated in this invention may be thosewhich are inflammatory disorders or are associated therewith and thosewhich may not be considered inflammatory disorders.

The methods and compositions of the present invention may be used inconjunction with other treatments, such as those for inflammatorydiseases. As demonstrated in Australian Patent Application No.2009201915 and in International Publication No. WO2010/020005, thecontents of which are incorporated herein by cross-reference,administration of a composition comprising adipose tissue-derived cells,which composition comprises adipocytes, directly into an joint affectedby inflammatory diseases, such as osteoarthritis, provides therapeuticbenefits for the patient and is associated with improvement in thejoint. The present invention additionally offers an adjunct treatment,whereby, for example, a treatment regime may be commenced byadministration of the composition comprising adipose tissue-derivedcells, which composition comprises adipocytes, into the affected joint,and that is followed by the less invasive course of treatment utilisingthe remote delivery of the present invention.

The present invention may also offer advantages as an adjunct treatmentfor arthritis and other inflammatory diseases in combination withestablished treatments. For example, a side-effect of long termtreatment of inflammatory conditions utilising NSAIDS is renal failure.The present invention offers an alternative to continued use of NSAIDSin patients with signs of renal failure. Alternatively, the use of thepresent invention in combination with NSAIDS may permit the NSAIDS to beused at a reduced dosage, thereby delaying the onset of renal failure.

The present invention may be used in combination with any therapy knownfor inflammatory diseases, including use with any of cartrophen,metacam, previcox, tramadol. The methods and compositions of the presentinvention may be used in conjunction with herbal medications, such asvalerian, rosemary oil and yucca leaves.

The present invention permits the preparation of cell secretions inadvance, such that a product comprising the adipose tissue-derived cellsecretions may be made available without the need to anesthetize asubject for extraction of adipose tissue. The present invention permitsthe preparation of compositions comprising (i) adipose tissue-derivedcell secretions, or (ii) an adipose tissue-derived cell suspension,optionally comprising adipocytes, or (iii) a combination of (i) and(ii), in advance of a treatment need. As described herein the cellsecretions or other compositions of the invention can be stored, forexample at −20° C. or below, until required for use.

Intensively Farmed Animals

As a result of intensive selection of farm animals over many generationsfor desirable traits, such as rapid growth, large muscle mass andefficient feed conversion in animals raised for meat production (eg.,pigs and cattle) and milk quality and volume in dairy animals, modernbreeds of farm animals often have a higher prevalence of susceptibilityto disease and acute and chronic conditions than do wild or lessintensively farmed populations. The incidence of and severity ofmanifestation of such diseases and conditions can be exacerbated by themanner in which an animal is farmed. Animals raised under intensivefarming conditions, such as in densely populated piggeries or in feedlots are typically susceptible to a higher incidence of disease andpotentially detrimental growth conditions than animals raised under lessintensive conditions, such as a free range or open grazing situation.Due to intensive selection, as described above however, even animalsraised in free range or open grazing operations may still have a highersusceptibility to or prevalence of detrimental health conditions. In thecontext of the invention therefore it will be understood that referenceto intensively farmed animals or intensive farming conditions includesintensively bred animals which may be raised in densely populatedoperations, for example with limited availability for individualmovement, and also includes intensively bred animals in less denselypopulated operations, such as where an individual has access to freerange or open grazing.

The identification by the inventor that the therapeutic compositions ofthe invention are effective when administered subcutaneously orintramuscularly is conducive to the methods and compositions of theinvention having beneficial effects in farming operations andparticularly intensive animal farming, such as intensive farming ofpigs, cattle, sheep and poultry. Whilst a therapeutic agent for alocalized disease, such as an arthritic joint, can be administered byintra-articular injection into the afflicted joint, such administrationrequires a high degree of skill on the part of the operator, as well aspotentially causing pain to the animal by handling of the afflictedjoint, and hence requiring restraint of the animal being treated. Incontrast, subcutaneous injection or intramuscular injection has a lowerskill requirement on the part of the operator, will generally notrequire handling of the afflicted limb, and typically requires onlyminimal restraint of the animal being treated. With the absence ofrequirement for high degree of operator skills, as compared tointra-articular injection for example, administration of thecompositions of the invention can be undertaken as part of a routineon-farm treatment or preventive program. In such an approach animals maybe administered the compositions of the invention at the same time asthe animals are being treated for other conditions, or at the same timeas the animals are undergoing routine procedures associated with theanimal breeding or growing enterprise, such as vaccination, ear tagging,tattooing, tail docking, or castration.

Generations of selectively breeding animals of a particular species fora desirable trait has contributed to the increased prevalence of certainundesirable characteristics in the population. As an example, pigs havefor many years been selected for rapid growth, large muscle mass andefficient feed conversion. This has, however, led to much greaterprevalence of skeletal, joint and cartilage problems than is seen in awild population. These problems can be exacerbated in a farmingoperation where the animal has limited room to move. Changes associatedwith cartilaginous structures may be referred to as leg weakness orosteochondrosis (OCD). The term “leg weakness” is also sometimes used todescribe poor leg conformation or to describe a clinical conditionassociated with lameness or stiffness. It may arise due to abnormalchanges in the articular cartilage and the growth (epiphyseal) plates,which are responsible for the growth of bones both in length anddiameter. The exact mechanisms that cause these changes are not fullyunderstood. They are thought to arise due to the pressure and sheerstresses that are placed upon these rapidly growing tissues, whichreduces oxygen supply causing abnormal growth and consistency of thecartilage. Reduced blood supply through a deficiency of blood vessel isalthough thought to contribute to such problems. Damage to the cartilagetends to be progressive and irreversible, the damaged cartilage beingreplaced by fibrous tissue.

Shortening and bending of the bones near the joints and at extremitiesof long bones may follow the cartilage damage. Weak epiphyseal platesalso have a tendency, to fracture and cartilage covering the jointsurfaces may split and form fissures. In modern pigs, such changes inthe cartilage take place from as early as two months of age. Thesepotentially detrimental changes are not generally clinically apparent atan early stage. In breeding enterprises it is not uncommon for 20% to30% of boars and gilts to be culled due to leg weakness and legdeformities. Such conditions, including OCD, thus have the potential forserious economic and ethical consequences for the fanning enterprise.There is currently no specific treatment for OCD. In advanced stages OCDmay also lead to arthritis and permanent lameness.

The compositions of the present invention are beneficial in the growthand repair and maturation of bone and cartilage. Accordingly, thepresent invention provides methods for treatment or prevention of boneand cartilage disorders in an animal, including leg weakness or OCD. AsOCD can lead to arthritis in an affected animal, the methods andcompositions of the invention can also be effective in preventing theoccurrence of or reducing the severity or incidence of arthritis in ananimal at risk of arthritis. In preferred embodiments the animal is apig. As described above, such conditions commence at an early age and,in early stages of development are typically not associated with anyclinical symptoms. In the methods of the invention the therapeuticcomposition may be administered to an animal which exhibits clinicalsymptoms of a joint disease or to an animal which does not exhibitclinical symptoms of a joint disease.

Clinical symptoms may include separation or fracture of the bones at theepiphyseal plate (epiphyseolysis) associated with sudden movement,lameness, sudden fractures (such as of the knee and elbow joints) whichmay be more common in young animals, abnormal leg conformation, abnormalgait, stiffness, pain. Age of visible onset of OCD can be variable, forexample within three months of gilts being introduced on to a farm,during the first pregnancy, in lactation or in the first two to threeweeks post weaning.

In an embodiment the invention provides a method for reducing theseverity of OCD in an animal, the method comprising administering tosaid animal a pharmaceutical composition comprising (i) adiposetissue-derived cell secretions, or (ii) an adipose tissue-derived cellsuspension, optionally comprising adipocytes, or (iii) a combination of(i) and (ii), wherein the administering is by subcutaneous injection orintramuscular injection. In preferred embodiments the animal is a pig.

Administration of the pharmaceutical composition to the recipient animalmay occur when the animal is at any suitable age. For prevention of theonset of clinical symptoms of OCD or for reducing the severity of OCD ina pig, the recipient is preferably treated at a young age. For example apig may be treated at, or soon after weaning, which typically occurs ina pig farming operation when the piglets are between about two and fiveweeks old. Young pigs may be treated at between about one month and sixmonths of age; or between about three months and six months of age. Inpiggeries, the pigs undergo a rapid growth phase at about 12 to 16 weeksof age. It is envisaged that in one embodiment the administration of thecompositions of the invention would be at the beginning of this growthperiod, for example at around 8 weeks of age, or 9 weeks of age, or 10weeks of age, or 11 weeks of age, or 12 weeks of age, or 13 weeks ofage.

The animal may receive a single administration of the composition of theinvention or may receive multiple administrations, such as two, three,four or more administrations. Where multiple administrations of thecomposition are performed, they will typically be separated by aboutone, two, three or four months in the case of administration to youngpigs.

The method is also beneficial in the treatment of symptomatic OCD. Insuch cases the recipient animal is typically an older animal, such asmore than six months old. In an older animals multiple administration ofthe compositions of the invention may be separated by about two to sixweeks, or by about one, two, three or four months or more. The choice oftiming of multiple administration may be determined by the skilledaddressee, for example it may be on the basis of re-appearance of orincreased severity of symptoms, pregnancy, weaning, etc.

As described above the compositions and methods of the invention findapplication in preventing onset of OCD in pigs and in reducing theseverity of OCD and other developmental orthopaedic conditions in pigs.Other mammals including humans, dogs, cattle, and horses, may alsosuffer developmental orthopaedic conditions, such as OCD, for examplethrough predisposition due to environment and or genetics. The inventionalso finds application in preventing onset of, or the severity of, or intreating, developmental orthopaedic conditions, such as OCD, in suchanimals.

As described herein the compositions of the invention are also capableof treating joint disease, arthritis and inflammatory conditions inafflicted mammals. The compositions of the invention are also useful inalleviating pain in mammalian subjects. The mammal may be any mammalsuch as a human, a domestic animal, a farm animal, such as a stud,breeder or grower animal, such as for meat or dairy production. In thetreatment of clinically relevant arthritis, for example, the farm animalmay be a pig. The pig may be a young pig or may be a breeder sow. Forexample a clinically affected pregnant sow may be treated to assist themto get through to farrowing.

Treatment of Pain

The inventor has identified that compositions of the invention areuseful in the treatment of subjects having pain. Various specific typesof pain are described herein, including the following.

Painful Musculoskeletal Conditions Other than Arthritis.

Painful musculoskeletal conditions are common, with a prevalence ofapproximately 30%. For musculoskeletal conditions other than arthritis,the prognosis is generally good; most people recover within a few weeksfollowing the onset of symptoms. However a significant minority do notrecover and develop long lasting or chronic pain, defined as painlasting longer than 3 months. For these patients prognosis is poor andrecovery is slow. A major focus of contemporary research is the earlyidentification of patients who are at a high risk of a poor outcome. Acommon finding from this research is that early high pain intensity is arisk factor for delayed recovery and the development of chronic pain.

The most frequent cause of chronic musculoskeletal pain is low backpain. Over 1 million Australians have a disability associated with theirback problems and it is the leading reason for Australians leaving theworkforce. Other common but less prevalent chronic pain conditionsinclude: neck and shoulder pain, whiplash associated disorder (WAD) andcomplex regional pain syndrome (CRPS).

Back pain is an extremely common, difficult-to-manage and expensivehealth condition. In Australia back pain is associated with costs ofaround $9 billion/year. Over 85% of low back pain is ‘non-specific’ lowback pain (NSLBP) in that a structural source of the pain cannot bereliably identified. Plausible therapeutic targets are innervatedtissues and include the disc, the facet joint and the sacroiliac joint,however other tissues such as muscle and ligament may also be involved.Up to 15% of low back pain is radiculopathy where impingement of thenerve root causes symptoms including back and leg pain. Pain associatedwith radiculopathy is believed to be associated with a localinflammatory process. Treatments for radicular pain include advice tostay active, analgesia including NSAIDS, epidural corticosteroidinjection and transfroaminal peri-radicular injections ofcorticosteroid.

Neck and Shoulder Pain

Whiplash associated disorder (WAD) are injuries to the neck caused byacceleration-deceleration energy transfer resulting most commonly from amotor vehicle accident. As with most painful musculoskeletal conditionsprognosis for new injuries is typically good in the first few weeks ormonths, but after 3 months recovery rates slow markedly and asignificant proportion of patients develops chronic Whiplash AssociatedDisorder (WAD). High pain intensity is known to be a predictor or pooroutcome. Whiplash is largely resistant to conservative treatments.

Complex Regional Pain Syndrome

Complex regional pain syndrome (CRPS) is a complication of minor trauma,usually to one limb, characterised by incapacitating pain, swelling,colour and temperature changes, and bone demineralisation in the limb.In Australia on average 5000 people are diagnosed with CRPS every year.The most common inciting minor trauma is wrist fracture. The incidenceof CRPS following wrist fracture is 5-7%. Prognosis is poor andtreatment options are often highly invasive, have significant sideeffect profiles and are only moderately effective. The major cause ofCRPS is thought to be an aberrant inflammatory response to tissue injuryand a more pro-inflammatory balance of inflammatory mediators isexpressed by patients with CRPS compared to those without the condition.

Neuropathic Pain

The inventor has identified that compositions of the invention areuseful in the treatment of subjects having pain for which there is nodiscernable clinical cause, such as some forms of neuropathic pain.Neuropathic pain refers to a group of painful disorders characterized bypain due to dysfunction or disease of the nervous system at a peripherallevel, a central level, or both. It is a complex entity with manysymptoms and signs that fluctuate in number and intensity over time. Thethree common components of neuropathic pain are steady and neuralgicpain; paroxysmal spontaneous attacks; and hypersensitivity.

Neuropathic pain can be very disabling, severe and intractable, causingdistress and suffering for individuals, including dysaesthesia andparaesthesia. Sensory deficits, such as partial or complex loss ofsensation, are also commonly seen. In addition, there are significantpsychological and social consequences linked to chronic neuropathicpain, which contribute to a reduction in quality of life.

Neuropathic pain is quite common in general medical practice. In someforms, the neuropathic pain is not associated with any discernableclinical causative condition. As an example it is demonstrated hereinthat the compositions of the invention are effective in alleviatingneuropathic facial pain. In some forms, the neuropathic pain isassociated with a discernable clinical condition. The prevalence oftrigeminal neuralgia is 2.1 to 4.7 persons per 100,000 of thepopulation, and of painful diabetic neuropathy occurs in 11% to 16% ofType 1 diabetics as well as Type II diabetics and postherpetic neuralgiais found in approximately 34 persons per 100,000 of the population.Treatment of neuropathic pain is not easy. Patients with neuropathicpain do not always respond to standard analgesics such as non-steroidalanti-inflammatory drugs (NSAIDs) and to some extent neuropathic pain isresistant to opiates. The pharmacologic agents best studied and longestused for the treatment of neuropathic pain are antidepressants andanticonvulsants both of which may have serious side effects.

A composition of the invention may be administered to a subject fortreatment of such pain at any appropriate site. Administration maytypically be using an appropriate type of injection or it may be bytopical application. For example, an injection may be subcutaneous,intramuscular, or directly into an accessible site at or near a site ofthe pain. As this type of pain may manifest in multiple areas of thesubject's body, for example jaw pain and limb or shoulder pain, theadministration may be at or near to one site of the pain and remote fromanother site afflicted by pain. Typically, where multiple sites of thepain occur in a patient, the administration is at or near a siteidentified as an original or primary site of the pain. As anillustration of this treatment, the examples herein show treatment ofneuropathic facial pain by injection into the subject's gum. A subjectbeing treated may be administered a single application of a compositionof the invention, such as a single injection or may be administeredmultiple applications, such as multiple injections.

The invention will now be described in more detail, by way ofillustration only, with respect to the following examples. The examplesare intended to serve to illustrate this invention and should not beconstrued as limiting the generality of the disclosure of thedescription throughout this specification.

EXAMPLES Example 1 Preparation of a Cell Free Extract

Preparation of Adipose Tissue

A 10 g sample of adipose tissue was collected by excision from theinguinal fat pad of a dog. The adipose tissue was rinsed with saline andthen minced finely using scissors and mixed with 20 ml of Dulbecco'sModified Eagle's Medium (DMEM, Sigma). Collagenase (Sigma) was added toproduce a final concentration of 0.05% w/v and the sample was incubatedat 37° C. for 30 minutes. At the end of 30 minutes the adipose tissuewas partially digested and consisted of a mixture of partially digestedfat particles, liberated stromal vascular cells (SVCs) and liberatedadipocytes. The sample was then centrifuged at 500 g for 15 minutes.Four distinct layers were visible within the centrifuged sample: a small(2 mm thick) layer of free lipid on the surface, below which was a white20 mm thick layer of adipose tissue and adipocytes and then a largeclear layer of DMEM and then a pellet of adipose tissue-derivednon-adipocyte cells. The small layer of lipid was carefully removed witha pasteur pipette. A fresh pasteur, pipette was then carefully insertedthrough the adipocytes and the clear DMEM was removed without disturbingthe floating adipose tissue, adipocytes or the pelleted cells. Thisresulted in a sample that contained only the floating pieces of adiposetissue and adipocytes suspended in a small volume of DMEM and thepelleted cells. The pieces of adipose tissue and adipocytes and thepelleted cells were gently mixed with a pasteur pipette and transferredto a 15 ml centrifuge tube. The pieces of adipose tissue and cells werethen washed in DMEM to remove collagenase as follows. DMEM was added toa final volume of 14 ml and the sample centrifuged at 500 g for 10minutes. This resulted in three distinct layers: floating pieces ofadipose tissue and adipocytes, DMEM and pelleted adipose tissue-derivednon-adipocyte cells. The DMEM was carefully removed by inserting apasteur pipette through the adipocytes taking care not to disturb thepieces of adipose tissue, adipocytes or the pelleted cells.

Tissue Culture

The floating pieces of adipose tissue and adipocytes and the pelletedcells were gently resuspended in 10 ml of DMEM and transferred to a 300ml tissue culture flask. A 30 ml volume of DMEM and 10 mls of autologoussterile serum were added and the flask was then incubated at 37° C. with5% CO₂. The flask was examined daily by microscopy. Cells becameattached and fibroblast-like in appearance between days 3 and 6. Theattached cells became confluent between days 5 and 10.

Harvesting Cell Free Cell Secretions

Once cells were confluent on the base of the flask the supernatant washarvested and the suspended adipose tissue and cells were removed byfiltration through a 20 micron mesh. The solution was filter sterilisedthrough a 0.22 micron filter and then aseptically dispensed into 10 mlvials and stored frozen at −20°. In the Examples that follow, thismaterial may be referred to as “CellFree”.

Example 2 Production of Concentrated Canine Cell Free Extracts

Volumes (100 ml) of the frozen cell free extracts in Example 1 werefreeze dried in a Telstar Lyobeta freeze dryer for 2 days. The resultingfreeze-dried cake was rehydrated with 10 ml of distilled water. Theconcentrated sample was then sonicated in a sonicating water bath for 20min. The 10 ml volumes contained a concentrated mix of cytokines.

Example 3 Subcutaneous Administration of Cell Free Extracts from CanineAdipose Tissue to Arthritic Dogs

In this example the safety and efficacy of subcutaneous injections ofthe canine cellular secretions prepared according to Example 1 above, inthe treatment of osteoarthritis in dogs was investigated. Five dogs eachreceived subcutaneous injections in the scruff of the neck once per weekfor four weeks at the rate of 0.3 ml/10 kg of body weight. The dogs inthis trial were predominantly advanced in age (>10 years) and had arange of joint and or bone disorders, including osteoarthritis of theelbow, chronic hip dysplasia, stifle disease and wobblers. Individuallythe dogs in this trial were each on a range of medications for thetreatment of their conditions (Table 1). The existing medication wasstopped whilst the trial was conducted. The dogs were assessed by theirregular treating vets, as well as being observed by their owners foranecdotal changes, such as changes in behaviour, mobility, and obvioussigns of pain. The trial is summarised in Table 1.

TABLE 1 Subcutaneous administration of cell free extracts from canineadipose tissue to arthritic dogs Continued Existing or stopped AdverseDog/Age Condition medication medication effects Comments Smudge ChronicOA Monthly Stopped None OA did not 12 y of elbow cartrophen observeddeteriorate Oz 14 y Chronic hip Monthly Stopped None OA did notdysplasia cartrophen observed deteriorate Daisy Stifle disease MetacamStopped None Marked 13 y observed improvement; less pain; more active;owners report better than Metacam Lassie Mild elbow None n/a NoneModerately 10 y OA observed less lame, more mobile; less pained VersaceWobblers Prednisone Stopped None Large 10 y observed improvement instability and activity levels

No adverse reactions were noted with repeated injections. All five dogsdid not deteriorate even though their current medications were stopped.Three of the five dogs showed significant improvement above what wasseen from their previous medications.

Example 4 Subcutaneous Administration of Concentrated Secretions fromCanine Adipose Tissue to Arthritic Dogs

The results of the trials presented in Example 3 demonstrated that thereare no adverse effects associated with the method of the invention andalso demonstrated that in some treated subjects the remoteadministration of the adipose tissue-derived cell secretions wasassociated with improved activity of the subject, improved mobility oran apparently lower degree of pain. The inventor reasoned thatadministration of a higher dose of adipose tissue-derived cellsecretions may provide further advantages.

A concentrated cell free Extract produced as described in Example 1 and2 was used to treat 9 arthritic dogs. A 2 ml volume of the concentratedcell free, extract was administered by subcutaneous injection into thescruff of the neck. The dogs were re-examined after 10 days by aveterinarian. Results are presented in Table 2.

TABLE 2 Subcutaneous administration of concentrated secretions fromcanine adipose tissue to arthritic dogs Breed Age (yrs) Condition Datetreated Outcome German 10 OA hips 5 Aug. 2011 Improved mobility and lessShepherd pain. Samoyed 11 Lameness 19 Jul. 2011 Improved mobility andless pain. Doberman 10 Wobblers 7 Jul. 2011 Great improvement. Dog morestable, active and has greater control over bowel movements. Border 12OA 5 Aug. 2011 More active, improved mobility Collie and less pain.German 6 OA 30 Jul. 2011 Much better. Shepherd More active, improvedmobility and less pain. Dogue de 3 OA 11 Jun. 2011 More active, improvedmobility bordeaux and less pain. Labrador 12 OA, 8 Aug . 2011 Moreactive, improved mobility neurological and less pain. issues. Bull 13Arthritic 1 Apr. 2011 More active and mobile, pain mastiff hips free.Effect has not waned cross when assessed after 5 months. (Chief) Golden12 Arthritic 28 Apr. 2011 More active and mobile, pain Retriever elbowsand free. Effect lasted 4 months. (Ruby) hips Effect was again apparentafter re-treatment.

Example 5 Subcutaneous Administration of Cell Free Extracts from CanineAdipose Tissue to Dogs with Atopic Dermatitis

A cell free extract prepared as described in Example 1 was administeredby subcutaneous injection to three dogs with atopic dermatitis. A 3 mlvolume was injected into the scruff of the neck, once a week for 3weeks. All three dogs showed an improvement when assessed by veterinaryexamination between 2 and 4 weeks after treatment, their condition wasimproved, with reduced inflammation being the primary outcome (Table 3).

TABLE 3 Subcutaneous administration of cell free extracts from canineadipose tissue to dogs with atopic dermatitis. Condition Name Age Breedtreated Outcome Comments Gemma 10 years  Bull mastiff x Atopic ModerateGenerally much less dermatitis improvement in inflamed, some skincondition papules still present; Topaz 6 years German Atopic MinorLesions on ventrum shepherd dermatitis improvement in less inflamed,scabby skin tail lesion dryer. BJ 2 years Kelpie Atopic Significant Feetlook great, left cross dermatitis improvement in arm lesion much skincondition improved, coat generally looks shinier. Original inflammationalong ventrum, arm, chest and paws improved.

Example 6 Subcutaneous Administration of Concentrated Cell Free Extractsfrom Equine Adipose Tissue in a Lame Horse

An equine cell free extract was prepared as described in Example 1 usingadipose tissue collected from the tail base of a horse. The adiposetissue was processed exactly as described in Example 1 except thatautologous equine serum was added to the tissue culture flask instead ofcanine serum. The cell free extract was concentrated by freeze drying asdescribed in Example 2.

A 2 ml volume of the concentrated cell free extract was administered bysubcutaneous injection into the neck of a racehorse that was mildlylame. The horse had previously been treated with intra-articularinjections of steroids and was no longer responding to this treatment.

The horse showed a marked reduction in lameness two days afteradministration of the cell free extract.

Example 7 Subcutaneous Administration of Concentrated Cell Free Extractsfrom Equine Adipose Tissue in Horses

Five horses with either suspensory ligament damage or early stageosteoarthritis of the knees were treated with concentrated equinesecretions (Example 6) by subcutaneous injection into the neck two daysbefore racing. All horses demonstrated improved mobility (Table 4).

TABLE 4 Subcutaneous administration of concentrated cell free extractsfrom equine adipose tissue in horses Date Problem Effect 16 Aug. 2011Early stage OA in knees Improved mobility and less lame, performedbetter than expected in race. 19 Aug. 2011 Suspensory ligament Improvedmobility and less lame, injury performed better than expected in race(won). 19 Aug. 2011 Suspensory ligament injury Improved mobility andless lame. 10 Sep. 2011 Suspensory ligament Improved mobility and lesslame, injury performed better than expected in race. 10 Sep. 2011 Earlystage OA in knees Improved mobility and less lame, performed better thanexpected in race.

Example 8 Treatment of Osteoarthritis in Dogs by Subcutaneous Injectionof Cryopreserved Allogeneic Adipose Derived Cells Including Adipocytes

Processing of Adipose Tissue

A 10 g sample of falciform adipose tissue was collected from a femaledog during a routine desex procedure. The adipose tissue was rinsed withsaline and then minced finely using scissors and mixed with 20 mls ofDulbecco's Modified Eagle's Medium (DMEM, Sigma). Collagenase (Sigma)was added to a final concentration of 0.05% (w/v) and the sample wasincubated at 37° C. for 90 minutes. During the incubation the sample wasgently inverted by hand every 15 minutes.

Following collagenase treatment the sample was aseptically filteredthrough a stainless steel mesh (700 micron pore size), transferred to a50 ml centrifuge tube and centrifuged at 500 g for 15 minutes.

Four distinct layers were visible within the centrifuged sample: a small(2 mm thick) layer of free lipid on the surface, below which was a white10 mm thick layer of adipocytes and then a large clear layer of DMEM andthen a pellet of SVF cells. The small layer of oil was carefully removedwith a pasteur pipette. A fresh pasteur pipette was then carefullyinserted through the adipocytes and the clear DMEM was removed withoutdisturbing the floating adipocytes or the pelleted SVF cells. Thisresulted in a sample that contained only the floating adipocytes and thepelleted SVF cells. The floating and the pelleted cells were gentlymixed with a pasteur pipette and transferred to a 15 ml centrifuge tube.

The cells were/then washed in DMEM to remove collagenase. DMEM was addedto a final volume of 14 mls and the sample centrifuged at 500 g for 10minutes. This resulted in three distinct layers: floating adipocytes,DMEM and pelleted SVF cells. The DMEM was carefully removed by insertinga pasteur pipette through the adipocytes taking care not to disturb theadipocytes or the pelleted cells.

The floating and the pelleted cells were gently resuspended in 4 mls ofDMEM and mixed with a pasteur pipette.

Expansion and Cryopreservation of Cells

Aliquots (0.5 mls) of the cell suspension were transferred to a T175tissue culture flask containing 50 mls of DMEM plus 10% canine serum andincubated in a CO₂ incubator at 37° C. until a confluent cell monolayerwas present (6 days). The floating cells (adipocytes) still had ahealthy morphology at this time.

Cells were stripped with 3 mls of TrypLE Express (Invitrogen), decantedinto 50 ml centrifuge tubes and centrifuged at 500×g for 10 minutes. Thefloating and the pelleted cells were resuspended in 2 mls of canineserum plus 10% DMSO and transferred to a cryovial. The cryovials werefrozen in a Mr Frosty slow freezing device (Invitrogen) in a −80° C.freezer for 24 hours and then transferred to a liquid nitrogen dewar.

Administration of Cells to Dogs

Seven dogs with osteoarthritis were given a single subcutaneousinjection of cells into the scruff of the neck. The cryovial per dog wasthawed at room temperature and the cell suspension drawn up with asyringe and hypodermic needle. The entire 2 mls of cell suspension wasinjected.

Monitoring of Dogs

Dogs were monitored over an 8 week period by their owners and wereexamined by a veterinarian. Six of the seven dogs showed a markedimprovement in their mobility and an apparent reduction in pain. Theseventh dog did not improve but did not show a negative response.

Example 9 Treatment of Osteoarthritis in Dogs by Subcutaneous Injectionof Cells Mixed with Cell Secretions

A vial of the cryopreserved cells as described in Example 8 was removedfrom liquid nitrogen and mixed with 5 mls of warm (37° C.) caninesecretions prepared as in Example 1. The cells and the secretions werethen drawn up into a syringe and administered subcutaneously into thescruff of a dog with arthritis. The dog responded well to the treatmentand showed a rapid improvement in mobility and a reduction in pain.

Example 10 Preparation of Adipose Derived Cells that had been Premixedwith Cell Secretions and then Frozen

Processing of Adipose Tissue

A 10 g sample of falciform adipose tissue was collected from a femaledog during a routine desex procedure. The adipose tissue was rinsed withsaline and then minced finely using scissors and mixed with 20 mls ofDulbecco's Modified Eagle's Medium (DMEM, Sigma). Collagenase (Sigma)was added to a final concentration of 0.05% w/v and the sample wasincubated at 37° C. for 90 minutes. During the incubation the sample wasgently inverted by hand every 15 minutes.

Following collagenase treatment the sample was aseptically filteredthrough a stainless steel mesh (700 micron pore size), transferred to a50 ml centrifuge tube and centrifuged at 500 g for 15 minutes.

The floating cells and the supernatant were discarded and the pelletedcells were gently mixed with a pasteur pipette and transferred to a 15ml centrifuge tube.

The cells were then washed in DMEM to remove collagenase. DMEM was addedto a final volume of 14 mls and the sample centrifuged at 500 g for 10minutes. The supernatant was discarded and the pelleted SVF cells weregently resuspended in 4 mls of DMEM and mixed with a pasteur pipette.

Expansion of Cells

Aliquots (0.5 mls) of the cell suspension were transferred to tissueculture flasks containing DMEM plus 10% canine serum and incubated in aCO2 incubator at 37° C. until a confluent cell monolayer was present (7to 10 days). Cells were stripped with 3 mls of TrypLE Express(Invitrogen), decanted into 50 ml centrifuge tubes and centrifuged at500×g for 10 minutes. Cells were either cryopreserved at this point, ina Mr Frosty slow freezing device as described in Example 8, or they wereplaced into new tissue culture flasks and passaged further until theyhad doubled approximately 8 or 13 times. The passaged cells were thenstripped and centrifuged.

Cryopreservation of Cells

The pelleted cell samples (no passage, approx. 8 doublings and approx.13 doublings) were each divided into two samples, one was mixed withconcentrated secretions whereas the other was not. The cells wereresuspended in either canine serum or a mixture of canine serum plusconcentrated canine secretions that were produced according to Example 1but that had been concentrated ten-fold by centrifuging in a 3 kDaAmicon centrifugal filter tube (Millipore). The concentrated secretionswere mixed with canine serum at a ratio of either 1 to 1 or at a ratioof 1 part concentrated secretions to 10 parts serum. The cell suspensionwere mixed with the serum and secretions and then held at roomtemperature for 30 minutes to allow the secretions to interact with thecells. The cell suspensions were then transferred to cryovials in 2 mlaliquots.

DMSO was added to each cryovial to produce a final concentration of 10%and the cryovials were frozen in a Mr Frosty slow freezing device(Invitrogen) in a −80° C. freezer for 24 hours and then transferred to aliquid nitrogen dewar for long term storage.

Thawing Cryopreserved Cells and Analysis of Cell Viability

Vials were removed from liquid nitrogen and allowed to thaw at roomtemperature. The vials were mixed by gently inverting and then a 0.1 mlvolume was removed for viability measurement. The 0.1 ml volume wasplaced in a flow cytometry tube and mixed with 0.9 mls of Isoflow(Beckman Coulter) that contained propidium iodide (Sigma ChemicalCompany, Louisville, USA) at a concentration of 10 μg/mL and Syto11(Molecular Probes, Eugene, USA) at a concentration of 1 μg/mL. Sampleswere analysed on a FACSCan flow cytometer and the percentage of Syto11positive (live cells) and propidium iodide positive (dead cells)recorded.

Culture of Cryopreserved Cells

A 1 ml volume of the thawed samples were placed into a T75 tissueculture flask with DMEM and 10% fetal calf serum and incubated at 37° C.with 5% in a CO₂ incubator for 5 days. Flasks were then examined eachday using an inverted microscope and the percentage confluency recorded.

Samples of tissue culture media from the flasks were removed andanalysed using the Bio-Plex Pro Human 27-plex cytokine, chemokine andgrowth factor assay (Biorad, Hercules, USA) for ILR1a, G-CSF, VEGF andIL-10. Control samples of fresh media with and without secretions wereincluded as controls.

Assessment of Cell Viability after Cryopreservation

The viability of cells frozen with the secretions was higher than thecells frozen without secretions (Table 5).

TABLE 5 Viability of passaged (13 cell doublings) and non-passaged cellsfrozen with and without secretions Cell type Average viabilitypost-thawing Non-passaged cells with no secretions 66.83% Non-passagedcells with secretions 75.70% Passaged cells (13 cell doublings) with35.95% no secretions Passaged cells (13 cell doublings) with  71.6%secretionsAssessment of Proliferation after Freezing Using the Click-iT EDU Assay

The proliferation rates of thawed cells were assessed using the Click-iTEDU assay (Life Technologies). A volume of the thawed cell suspensionthat contained approximately 500,000 cells was transferred to a 6 wellplate. A 2 ml volume of DMEM plus 10% canine serum was added to thewell. The EDU reagent was added to the well to create a finalconcentration of 10 μM. The plate was incubated at 37° C. with 5% CO₂ ina CO2 incubator at 37° C. for 2 hours. The supernatant was collected andthe adherent cells were stripped and both were combined, washed (in PBSplus 1% bovine serum albumin; Sigma) and resuspended in 100 μL ofClick-iT fixative and incubated for 15 min at room temperature to fixthe cells. Cells were then washed and resuspended in 100 μL of Click-iTpermeabilisation agent to permeabilise cells. The cells were labelledfor detection, by is incubating (30 min) in the Click-iT reactioncocktail (500 μL) containing 2.5 μL of AlexaFluor 488 Fluorescent dye.Following incubation the cells were washed in Click-iT permeabilisationand wash reagent and was analysed by flow cytometry. Cells were thenanalysed using a FACScan flow cytometer. Proliferating cells showed anincrease in green fluorescence compared to non-proliferating cells.

The results of analysing the cells are displayed in FIG. 1. The samplethat had been stored frozen as cells plus secretions showed a populationof proliferating cells equal to 16.8% of the total cells. The samplesthat had been stored frozen as cells with no secretions showed apopulation of proliferating cells equal to only 1.8% of the total cells.

Recovery of Cells after Freezing

The addition of secretions to cells prior to freezing the cells improvedthe ability of the cells to survive the freezing process and toproliferate after thawing. Cells that had been cultured until theyreached cumulative cell doublings of approximately 8 and that had beenfrozen with secretions were observed to attach to the tissue cultureflask more rapidly and grow more rapidly than the same cells frozenwithout secretions. At 24 hours the cells frozen with secretions hadreached 90% confluency whereas the cells frozen without secretions hadreached only 60% confluency (Table 6).

TABLE 6 Assessment of cultured cells (approximately 8 cumulative celldoublings) frozen with and without secretions Confluency after 24 hoursin culture Cell type post-thaw Cells frozen with no secretions 60% Cellsfrozen with secretions 90%Comparison of Cytokines Produced from Cells Frozen with and withoutSecretions

Cells that had been cultured through approximately 8 cumulative celldoublings and that were frozen with secretions produced a statisticallysignificant (two-tailed t-test) greater amount of the cytokines ILr1a,IL-6, IL-8, IL-9, IL-12, IL-13, FGF basic, TNF-a and VEGF than the samecells frozen without secretions (Table 7), upon post-thaw culturing for5 days.

TABLE 7 Comparison of cytokines produced by cells that had been frozenwith or without secretions. The numbers are the means of threereplicates. Sample IL-1ra IL-6 IL-8 IL-9 IL-10 IL-12 IL-13 FGF basicTNF-a VEGF Cells with 80 8 17 10 43 91 8 14 20 5166 secretions Cells 200 0 0 18 38 3 0 1 988 p-value 0.005 0.000 0.006 0.000 0.115 0.016 0.0390.000 0.001 0.059

These sets of data demonstrate that there is a beneficial effect ofcombining secretions with cells prior to freezing the cells. Cells thatare frozen without secretions lose their ability to proliferate and toproduce cytokines. Including secretions with the cells prior to freezingpreserves the ability of the cells to proliferate and to producecytokines.

Example 11 Production of Secretions from Passaged Cells and Use of theSecretions to Freeze Cells

Canine adipose derived cells were isolated and cultured as described inExample 10. The cells were passaged until the cells had reached acumulative cell doubling of approximately 13 times. The tissue culturesupernatant from the cells was concentrated using a 3 kDa Amiconcentrifugal filter tube (Millipore). The concentrated secretions weremixed with serum at a ratio of 1 to 1. The cells were stripped, washedand the cell pellet was resuspended in the mixture of serum andsecretions and then held at room temperature for 30 minutes to allow thesecretions to interact with the cells. The cell suspensions were thentransferred to cryovials, mixed with 10% DMSO and frozen as described inExample 10.

The cryopreserved cell suspensions were thawed and cultured in tissueculture flasks as described in Example 10. At 72 hours the cells frozenwith secretions had reached 90% confluency whereas the cells frozenwithout secretions had reached only 20% confluency (FIG. 2).Importantly, the cells without secretions became senescent and did notreach greater than 20% confluency even after 10 days of incubation.

This is an important finding. There is a need to be able to expandmesenchymal stem cells and other cell types in tissue culture to producelarge numbers of cells. However, mesenchymal stem cells and other celltypes cannot be cultured indefinitely. After a number of cell doublingsthe cells become senescent and will not multiply any further. Thislimits the number of doses that can be produced from a single culture.

To produce large numbers of cells from a single starting culturerequires a cell bank of cryopreserved vials to be set up. Normally adouble cell bank is created. A first set of cryopreserved vials iscreated and one of these vials is then thawed and used to produce asecond set of cryopreserved vials. Each time a batch of product isproduced one of these second set of vials is thawed and placed intoculture. This cell banking process typically results in cells that arenot as therapeutically effective as freshly isolated cells. By combiningsecretions with the cells during the freezing steps overcomes thisproblem and allows the production of a therapeutically effective frozenproduct.

Example 12 Treatment of Neuropathic Facial Pain with Autologous AdiposeDerived Cells

Four human patients suffering from neuropathic facial pain were treatedwith an autologous adipose derived cell suspension comprised of stromalvascular fraction cells and adipocytes.

Liposuction was used to collect approximately 200 grams of adiposetissue from the abdomen and or thighs of each patient. The lipoaspiratewas processed immediately after collection by washing with sterilesaline and then digesting by adding sterile collagenase to a finalconcentration of 0.05% w/v. The sample was incubated at 37° C. for 20minutes, filtered through a 800 micron mesh and transferred tocentrifuge tubes.

The centrifuge tubes were centrifuged at 400 g for 10 minutes, and thelayer between the floating adipocytes and the pelleted cells wasremoved. The cell pellet and floating adipocytes were combined andfilter sterilized saline was added until the tubes were full. Thesamples were centrifuged again at 400 g for 10 minutes and the layerbetween the pelleted cells and the floating adipocytes was removed. Theresulting cell preparation was diluted to a volume of 10 ml with sterilesaline and dispensed into 2 ml volumes in sterile syringes.

The patients were given four or five injections of 2 mL volume intotheir gums at the original site of the pain.

Patients were followed up at between 4 and 8 weeks post treatment andtheir level of pain was assessed using a visual analogue scale (0=nopain, 10=worst pain imaginable). The results from these assessments aredescribed in the Table 8 below.

TABLE 8 Administration of autologous adipose derived cells for treatmentof neuropathic pain Pretreatment condition Post treatment conditionPatient 1 Constant sharp neuralgic At one month post treatment: pain allday, Two or three pain episodes each pain in tooth and cheek, day (2-3hours each), pain intensity 8. pain in tooth only, pain intensity 6-7.Patient 2 Pain in arm, shoulder, jaw,, At two months post treatment:teeth pain intensity 9. mild tenderness in tooth only, pain intensity0-1. Patient 3 Pain in teeth, jaw and gum, At 3 weeks post treatment:pain intensity 8, Neurontin has been reduced to needed Neurontin 1200 mg100 mg and pain in teeth and and Endep 50 m. gum only, pain intensity 4.Patient 4 Facial pain intensity 8-9, At one month post treatment: only 2hours sleep at night facial pain intensity 2 but and woken by sharppain. episode of higher pain intensity from cold weather, sleeps 6hours.

Example 13 Treatment of Pain with Topical Application of Secretions fromBovine Adipose Derived Cells

A 10 g sample of adipose tissue was collected by excision from thetail-base of a 1 year old steer. The adipose tissue was rinsed withsaline and then minced roughly using scissors into pieces ofapproximately 5 mm diameter and mixed with 20 ml of Dulbecco's ModifiedEagle's Medium (DMEM, Sigma). Collagenase (Sigma) was added to produce afinal concentration of 0.2% [w/v] and the sample was incubated at 37° C.for 30 minutes. At the end of 30 minutes the adipose tissue waspartially digested and consisted of a mixture of partially digested fatparticles, liberated stromal vascular cells (SVCs) and liberatedadipocytes.

The sample was then washed to remove collagenase by centrifuging at 500g for 15 minutes. Four distinct layers were visible within thecentrifuged sample: a small (2 mm thick) layer of free lipid on thesurface, below which was a white 20 mm thick layer of adipose tissue andadipocytes and then a large clear layer of DMEM/collagenase and then apellet of adipose tissue-derived non-adipocyte cells. The small layer oflipid was carefully removed with a pasteur pipette. A fresh pasteurpipette was then carefully inserted through the adipocytes and the clearDMEM was removed without disturbing the floating adipose tissue,adipocytes or the pelleted cells. This resulted in a sample thatcontained only the floating pieces of adipose tissue and adipocytessuspended in a small volume of DMEM and the pelleted cells. The piecesof adipose tissue and adipocytes and the pelleted cells were gentlymixed with a pasteur pipette and transferred to a 15 ml centrifuge tube.

The pieces of adipose tissue and cells were then washed in DMEM toremove collagenase as follows. DMEM was added to a final volume of 14 mland the sample centrifuged at 500 g for 10 minutes. This resulted inthree distinct layers: floating pieces of adipose tissue and adipocytes,DMEM and pelleted adipose tissue-derived non-adipocyte cells. The DMEMwas carefully removed by inserting a pasteur pipette through theadipocytes taking care not to disturb the pieces of adipose tissue,adipocytes or the pelleted cells.

Tissue Culture

The floating and the pelleted cells were gently resuspended in 10 ml ofDMEM and transferred to a 300 ml tissue culture flask. A 30 ml volume ofDMEM and 10 mls of sterile fetal calf serum were added and the flask wasthen incubated at 37° C. with 5% CO₂. The flask was examined daily bymicroscopy. Cells became attached and fibroblast-like in appearancebetween days 3 and 6.

Harvesting Cell Free Cell Secretions

After 6 days the supernatant was harvested and the suspended adiposetissue and cells were removed by filtration through a 20 micron mesh.The solution was filter sterilised through a 0.22 micron filter and thenaseptically dispensed into 10 ml vials and stored frozen at −20° C.

Preparation of Cream Containing Bovine Secretions and a Placebo Cream

A vial of the bovine secretions was thawed and mixed with an equalamount of Aqueous Base Cream BP and dispensed into plastic tubes andstored at 4° C. until used.

A second lot of cream that did not contain bovine secretions wasprepared as a placebo control. DMEM was mixed with an equal amount ofAqueous Base Cream BP and dispensed into plastic tubes.

Treatment of Pain from Burns

A 46 year old male who had accidentally mildly burnt his thumb andmiddle finger was given a tube of each cream. The tubes were labelled asCream 1 and Cream 2 and the man did not know which cream contained thesecretions or which was the placebo. The man applied Cream 1 to themiddle finger and Cream 2 to the thumb approximately 30 minutes afterthe burns occurred. The man reported that after 10 minutes there was nolonger any pain in his thumb. The pain in the thumb did not return. Thepain in the middle finger continued for approximately 6 hours. A blisterformed on the middle finger but no blister occurred on the thumb.

Cream 1 was the placebo and was applied to the middle finger. Cream 2contained the bovine secretions and was applied to the thumb.

Example 14 Intramuscular Administration of Secretions from HumanAdipose-derived Cells to Mice with Collagen Antibody-induced Arthritis

Preparation of Adipose Tissue

Liposuction was used to collect approximately 200 grams of adiposetissue from a patient. The lipoaspirate was digested by adding sterilecollagenase to a final concentration of 0.05% w/v. The sample wasincubated at 37° C. for 30 minutes, filtered through a 800 micron meshand transferred to centrifuge tubes. The tubes were centrifuged at 1500g for 5 minutes, to obtain the pelleted cells (stromal vascularfraction; SVF) and floating adipocytes. Four distinct layers werevisible within the centrifuged sample: a small layer of free lipid onthe surface, below which was a thick layer of adipose tissue andadipocytes and then a large clear layer of saline and a pellet ofadipose tissue-derived non-adipocyte cells (SVF). The lipid layer wasaspirated and discarded. The adipocyte and SVF fractions were separatelycollected after the centrifugation. The fractions were washed separatelywith saline and centrifuged at 1500×g for 5 mins. The SVF pellet wasgently resuspended in 10 mL of cell culture media that consisted ofDulbeccos Modified Eagle Medium (DMEM) supplemented with 10% foetalbovine serum and 1% penicillin-streptomycin solution.

A 300 μL portion of the SVF pellet was filtered through a 35 μm nylonmesh topped tube. A 200 μL portion of the filtered sample was enumeratedand the viability determined in TruCount tubes containing isoflow,propidium iodide (10 μg/mL) and Syto11 (1 μM) using a FacsScan flowcytometer. The total number of viable nucleated cells in the SVF pelletwas determined.

Tissue Culture and Harvesting of Secretions

A T175 cm² culture flask was seeded with approximately 29 million viableSVF cells and 30 mL of adipocytes. A volume of 50 mL of cell culturemedia, as described above, was also added to the flask. The flask wasincubated at 37° C. with 5% CO₂ for 72 hours.

Following the 72-hour incubation, the conditioned medium was collectedfrom the flask. This conditioned media sample was centrifuged at 4980 gfor 10 mins and stored at −80° C. This conditioned media was thawed,filter sterilized using 0.22 μm syringe filter, aliquoted and frozen at−80° C. A vehicle control, containing DMEM supplemented with 1%penicillin-streptomycin solution, was also filter sterilized using a0.22 micron syringe filter, aliquoted and frozen at −80° C. Thesealiquoted samples were given coded names and were shipped on dry ice toTetraQ where they were administered to mice suffering from CAIA.

Collagen Antibody-induced Arthritis Mouse Model

The collagen antibody-induced arthritis (CAIA) model is a widelyaccepted animal model of arthritis which has been reported in theliterature to investigate the pathogenic mechanisms involved inarthritis and to screen potential therapeutic candidates. The modeltargets type I collagen, one of the major constituents of articularcartilage. In mice, CAIA is induced by the administration of a cocktailof 5 anti-type II collagen antibodies followed by a lipopolysaccharide(LPS) injection three days later. The subsequent administration of LPSfollowing the antibody cocktail not only increases the severity of thearthritis through the induction of pro-inflammatory cytokines andcomplement component activation, but also reduces the amount ofmonoclonal antibody required to induce the arthritis in this model. Thearthritis that develops in these mice closely resembles rheumatoidarthritis in people, including synovitis with infiltration ofpolymorphonuclear and mononuclear cells, pannus formation, fibrosiscartilage degradation and bone erosion. Significant swelling and rednessis observed in the paws of mice suffering with CAIA. The clinicalmanifestations of paw redness and swelling can be assessed to assign aclinical arthritis score to mice in the CAIA model. Outcome measures ofpaw volume, ankle size and clinical arthritis score can be used todetermine the effectiveness of a treatment on reducing the severity ofarthritis in this CAIA model.

At day zero, each mouse (total of 12) received an intravenous injectionof 1.5 mg (150 μL) of an anti-type II collagen 5 clone antibodycocktail. This cocktail contains 5 monoclonal antibodies: Clone A2-10(IgG2a), F10-21 (IgG2a), D8-6 (IgG2a), D1-2G (IgG2b), and D2-112 (IgG2b)recognizing the conserved epitopes on various species of type IIcollagen. On day 3, mice received an intraperitoneal injection of 80 μL(40 μg/mouse) of LPS. The trial design consisted of 2 groups of 6 mice.The mice received 50 μL doses administered via the intramuscular (IM)route on days 6, 8, 10 and 12 of the following: group 1 received humansecretions from the SVF+adipocytes and group 2 received the vehiclecontrol.

The mice were monitored throughout the trial period (2 weeks) and theprimary outcome measurements of paw volume, ankle size and clinicalarthritis score were taken on days 0 and 2-13. The measurements weretaken prior to administration of the collagen antibody cocktail (days 0)and LPS (day 3) and prior to administration of vehicle and the humanSVF+adipocyte secretions. Paw volume was measured using aplethysmometer. The paw size was measured using microcalipers across thehillock (ankle joint) of each hindpaw. The mice were assessed and scoredfor the severity of arthritis using a standard scale (0—normal; 1—mildredness, slight swelling of ankle or wrist, redness and swelling limitedto individual joints; 2—moderate swelling of ankle or wrist, redness inmore than one joint, 3—severe swelling including some digits, ankle orfoot; 4—maximal swelling and inflamed, involving multiple joints).

Data Analysis

The average and standard deviation (SD) of each of the primary outcomemeasures, paw volume (cm³), ankle size (mm), and clinical arthritisscore were calculated for each time-point post-treatment for both groupsof mice. A two-tailed t-test was performed on the primary outcomemeasures at each time-point post-treatment to compare the effect of theSVF+adipocyte secretions versus the vehicle control. The statisticalsignificance criterion was P<0.05.

Results

The paw volume results for both groups are presented in FIG. 3. Ananalysis of the data at day 11 revealed a significant reduction(p-value=0.002) in the paw volume of mice treated with SVF+adipocytesecretions when compared to the vehicle control mice.

The ankle size measurements for both groups are presented in FIG. 4. Ananalysis of the data at day 11 revealed a significant reduction(p-value=0.018) in the ankle size of mice treated with SVF+adipocytesecretions when compared to the vehicle control mice.

The clinical arthritis scores for both treatment groups are presented inFIG. 5. An analysis of the data at day 11 revealed a significantreduction (p-value=0.017) in the clinical arthritis score of micetreated IM with SVF+adipocyte secretions when compared to the vehiclecontrol mice.

The data clearly shows that administration of secretions byintra-muscular injection has a therapeutic effect in the CAIA mousemodel. The injection site is remote from the site of the disease.

Example 15 Intravenous Administration of Adipose-derived CellsCryopreserved in Standard Cryoprotectants Versus Cells Cryopreservedwith Concentrated Secretions to Mice with Collagen Antibody-inducedArthritis

Preparation of Adipose Tissue

Canine adipose tissue was processed as described in Example 1 to producecanine adherent cells. Furthermore, canine secretions which wereconcentrated 10×, using a 3 kDa Amicon centrifugal filter tube, fromadipose-derived cells, were produced as described in Example 1. Fromthese cells and secretions, the following test products were prepared incryovials and cryopreserved in a Mr Frosty slow freezing device(Invitrogen) in a −80° C. freezer for 24 hours and then transferred to aliquid nitrogen dewar:

-   -   1. 70,000 cells cryopreserved in 90% canine serum and 10% DMSO    -   2. 70,000 cells cryopreserved in 45% canine secretions (10×        concentrate), 45% canine serum and 10% DMSO.        Collagen Antibody-induced Arthritis Mouse Model

The collagen antibody-induced arthritis mouse (CAIA) model described inExample 14 was also used to investigate the effects of administering theabove described test products. A total of 12 mice were induced with CAIAas described in Example 14. At day 6 post-CAIA induction, the abovedescribed cryopreserved test products were thawed at room temperatureimmediately before injection. 6 mice were each injected intravenouslywith 140 μL of cells (Which equated to 70,000 cells) which had beencryopreserved in standard cryoprotectants, and the remaining 6 mice wereeach injected intravenously with 140 μL of cells (which equated to70,000 cells) which had been cryopreserved in a cryoprotectant mixturewhich included concentrated secretions.

The mice were monitored daily throughout the trial and the primaryoutcome measures of paw volume, ankle size and clinical arthritis scorewere taken on days 0 and 2-12 as described in Example 14.

Data Analysis

The average and standard deviation (SD) of each of the primary outcomemeasures, paw volume (cm³), ankle size (mm), and clinical arthritisscore were calculated for each time-point post-treatment for both groupsof mice and graphed. The delta (Δ) paw volume, ankle size and clinicalarthritis score was calculated by subtracting the pre-CAIA inductionscore from the post-CAIA induction scores and expressing this as apercentage change. The area under the Δ paw volume, ankle size andclinical arthritis curves were determined for each group. A two-tailedt-test was used to compare the Δ paw volume, ankle size and clinicalarthritis area under the curve (AUC) values from both groups.

Results

The paw volume results for both treatment groups are presented in FIG.6. An area under the curve analysis of this data revealed a significantreduction in the paw volume of mice treated with cells and concentratedsecretions when compared to cells alone (FIG. 7).

The ankle size measurements for both treatment groups are presented inFIG. 8. A significant reduction in the ankle size of mice treated withcells and concentrated secretions was observed when compared to the micethat received cells only (FIG. 9)

The clinical arthritis scores for both treatment groups are presented inFIG. 10. A significant reduction in the clinical arthritis score wasobserved in the mice treated with cells and secretions when compared tomice that received cells alone (FIG. 11).

The data shows an increased therapeutic effect by combining cells withsecretions prior to freezing the cells. The data clearly shows that afrozen product that combines cells and secretions has a therapeuticeffect in the CAIA mouse model. When cells alone were administered therewas no therapeutic effect. This is due to the cells being damaged duringthe freezing process and the damaged cells are not secreting thecytokines that are required to cause the therapeutic effect. By freezingthe cells with secretions the cells are able to survive the freezingprocess and be fully functional and capable of secreting the cytokinesrequired to cause the therapeutic effect.

Example 16 Treatment of Pain in Dogs by Subcutaneous Administration ofAdipose Derived Cells

Frozen cells were prepared according to Example 10. The cell suspensionwas thawed and administered subcutaneously in the scruff of the neck ofa dog that was suffering from sciatic nerve pain. One week aftertreatment the dog showed no signs of pain.

A second dog that was suffering from intervertebral disc disease and wasadministered a subcutaneous injection of cells in to the scuff of theneck. One week after treatment the dog showed no signs of pain.

The invention claimed is:
 1. A method of alleviating pain in a mammalian subject, the method comprising, administering to the subject a pharmaceutical composition which comprises a combination of adipose tissue-derived cell secretions and multiply-passaged adherent cells from an adipose tissue-derived cell suspension, wherein said adipose tissue-derived cell secretions are prepared by culturing multiply-passaged adherent progeny cells from an adipose tissue-derived cell suspension and harvesting supernatant from the cell culture after about 3 or more days, said adherent progeny cells having a fibroblast-like appearance, and wherein said combination has been cryopreserved.
 2. The method according to claim 1, wherein said administration to said subject is by intra-articular injection or by intramuscular injection or by subcutaneous injection or by topical administration.
 3. The method according to claim 1, wherein the pain is back pain, or is pain associated with a burn injury, an inflammatory disorder, a ligament injury, a tendon injury, or the sciatic nerve, or is neuropathic pain.
 4. The method according to claim 3, wherein the neuropathic pain is facial pain, pain of the teeth, jaw, gum or of a limb.
 5. The method according to claim 1, wherein the adipose tissue-derived cell suspension (i) comprises adipocytes or (ii) is substantially free of adipocytes.
 6. The method according to claim 1, wherein the subject is selected from the group consisting of (i) a human, (ii) poultry, (iii) an equine animal, (iv) a feline animal, (v) a canine animal, (vi) a bovine animal, and (vii) a porcine animal.
 7. The method according to claim 1, wherein the adipose tissue-derived cell secretions or the adipose tissue-derived cell suspension is (i) derived from adipose tissue allogeneic to the recipient subject or animal, or (ii) derived from the intended recipient subject or animal, or (iii) derived from adipose tissue xenogeneic to the recipient subject or animal.
 8. The method according to claim 1, wherein the adipose tissue-derived cell secretions is a preparation concentrated by between 2-fold and 20-fold.
 9. The method according to claim 1, wherein said adipose tissue-derived cell secretions comprises concentrated media from culture of an adipose tissue-derived cell suspension, optionally wherein said adipose tissue-derived cell suspension (i) comprises adipocytes and adipose tissue-derived mesenchymal stem cells or (ii) is substantially free of adipocytes, optionally wherein said media from culture of an adipose tissue-derived cell suspension is concentrated between 2-fold and 20-fold.
 10. The method according to claim 1, wherein the method further comprises thawing the cryopreserved combination prior to administration to the recipient subject or animal.
 11. The method according to claim 10, wherein the cryopreserved combination, or pharmaceutical composition thereof, is administered to the recipient subject or animal within two hours of thawing.
 12. A kit comprising (a) a pharmaceutical composition comprising a combination of adipose tissue-derived cell secretions and multiply-passaged adherent cells from an adipose tissue-derived cell suspension, wherein said adipose tissue-derived cell secretions are prepared by culturing multiply-passaged adherent progeny cells from an adipose tissue-derived cell suspension and harvesting supernatant from the cell culture after about 3 or more days, said adherent progeny cells comprising cells having a fibroblast-like appearance, wherein said combination or composition is cryopreserved; and (b) instructions for use of said kit in alleviating pain in a mammalian subject.
 13. The kit according to claim 12, wherein the pain is back pain, or is pain associated with a burn injury, an inflammatory disorder, a ligament injury, a tendon injury, or the sciatic nerve, or is neuropathic pain, optionally wherein the neuropathic pain is facial pain, pain of the teeth, jaw, gum or of a limb.
 14. The kit according to any claim 12, wherein the adipose tissue-derived cell suspension (i) comprises adipocytes, or (ii) is substantially free of adipocytes.
 15. The kit according to claim 12, further comprising instructions for administration of said composition within 2 hours of thawing said cryopreserved composition.
 16. The kit according to claim 12, wherein the adipose tissue-derived cell secretions or the adipose tissue-derived cell suspension is (i) derived from adipose tissue allogeneic to the recipient subject or animal, or (ii) derived from the intended recipient subject or animal, or (iii) derived from adipose tissue xenogeneic to the recipient subject or animal.
 17. The kit according to claim 12, wherein the adipose tissue-derived cell secretions is a preparation concentrated by between 2-fold and 20-fold.
 18. The kit according to claim 12, wherein said adipose tissue-derived cell secretions comprises concentrated media from culture of an adipose tissue-derived cell suspension, optionally wherein said adipose tissue-derived cell suspension (i) comprises adipocytes and adipose tissue-derived mesenchymal stem cells or (ii) is substantially free of adipocytes, optionally wherein said media from culture of an adipose tissue-derived cell suspension is concentrated between 2-fold and 20-fold. 